Compositions comprising streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof

ABSTRACT

The invention is related to multivalent immunogenic compositions comprising more than one  S. pneumoniae  polysaccharide protein conjugates, wherein each of the conjugates comprises a polysaccharide from an  S. pneumoniae  serotype conjugated to a carrier protein, wherein the serotypes of  S. pneumoniae  are as defined herein. In some embodiments, at least one of the polysaccharide protein conjugates is formed by a conjugation reaction comprising an aprotic solvent. In further embodiments, each of the polysaccharide protein conjugates is formed by a conjugation reaction comprising an aprotic solvent. Also provided are methods for inducing a protective immune response in a human patient comprising administering the multivalent immunogenic compositions of the invention to the patient. The multivalent immunogenic compositions are useful for providing protection against  S. pneumoniae  infection and/or pneumococcal diseases caused by  S. pneumoniae . The compositions of the invention are also useful as part of treatment regimes that provide complementary protection for patients that have been vaccinated with a multivalent vaccine indicated for the prevention of pneumococcal disease.

FIELD OF THE INVENTION

The present invention provides multivalent immunogenic compositionshaving distinct polysaccharide-protein conjugates. Each conjugateconsists of a capsular polysaccharide prepared from a different serotypeof Streptococcus pneumoniae conjugated to a carrier protein, preferablyCRM197. The immunogenic compositions provide broad coverage againstpneumococcal disease.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 3, 2019, isnamed 24683USNP-SEQTXT-03DEC2019 and is 6 kilobytes in size.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is a Gram-positive bacterium and the mostcommon cause of invasive bacterial disease (such as pneumonia,bacteraemia, meningitis and Otitis media) in infants and young children.Pneumococcus is encapsulated with a chemically linked polysaccharidewhich confers serotype specificity. There are over 90 known serotypes ofpneumococci, and the capsule is the principle virulence determinant forpneumococci, as the capsule not only protects the inner surface of thebacteria from complement, but is itself poorly immunogenic.Polysaccharides are T-cell independent antigens, and, in most cases, cannot be processed or presented on MHC molecules to interact with T-cells.They can however, stimulate the immune system through an alternatemechanism which involves cross-linking of surface receptors on B cells.

The multivalent pneumococcal polysaccharide vaccines that have beenlicensed for many years have proved valuable in preventing pneumococcaldisease in adults, particularly, the elderly and those at high-risk.However, infants and young children respond poorly to unconjugatedpneumococcal polysaccharides. The pneumococcal conjugate vaccine,Prevnar®, containing the 7 most frequently isolated serotypes (4, 6B,9V, 14, 18C, 19F and 23F) causing invasive pneumococcal disease in youngchildren and infants at the time, was first licensed in the UnitedStates in February 2000. Following universal use of Prevnar® in theUnited States, there has been a significant reduction in invasivepneumococcal disease in children due to the serotypes present inPrevnar®. See Centers for Disease Control and Prevention, MMWR MorbMortal Wkly Rep 2005, 54(36):893-7. However, there are limitations inserotype coverage with Prevnar® in certain regions of the world and someevidence of certain emerging serotypes in the United States (forexample, 19A and others). See O'Brien et al., 2004, Am J Epidemiol159:634-44; Whitney et al., 2003, N Engl J Med 348:1737-46; Kyaw et al.,2006, N Engl J Med 354:1455-63; Hicks et al., 2007, J Infect Dis196:1346-54; Traore et al., 2009, Clin Infect Dis 48:S181-S189.

U.S. Patent Application Publication No. US 2006/0228380 describes a13-valent pneumococcal polysaccharide-protein conjugate vaccineincluding serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and23F. Chinese Patent Application Publication No. CN 101590224 A describesa 14-valent pneumococcal polysaccharide-protein conjugate vaccineincluding serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19Fand 23F.

Other PCVs have covered 7, 10, 11, or 13 of the serotypes contained inPCV-15 (U.S Pub. No. 2011/0195086), but immune interference has beenobserved for some serotypes (e.g. lower protection for serotype 3 inGSK's PCV-11) and lower response rates to serotype 6B in Pfizer's PCV-13(PREVNAR® 13). See Prymula et al., 2006, Lancet 367:740-48 and Kieningeret al., Safety and Immunologic Non-inferiority of 13-valent PneumococcalConjugate Vaccine Compared to 7-valent Pneumococcal Conjugate VaccineGiven as a 4-Dose Series in Healthy Infants and Toddlers, presented atthe 48^(th) Annual ICAAC/ISDA 46^(th) Annual Meeting, Washington D.C.,Oct. 25-28, 2008.

The current multivalent pneumococcal vaccines have been effective inreducing the incidence of pneumococcal disease associated with thoseserotypes present in the vaccines. However, the prevalence of thepneumococci expressing serotypes not present in the currently availablevaccines has been increasing. Accordingly, there is a need foradditional pneumococcal vaccine compositions which can provideprotection against pneumococcal serotypes not present in currentlyavailable vaccines.

SUMMARY OF THE INVENTION

The invention provides multivalent immunogenic compositions comprisingS. pneumoniae polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, and wherein the polysaccharide proteinconjugates include polysaccharides of a group of S. pneumoniae serotypesselected from the group consisting of:

-   -   a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A,        DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; and aa) 1, 3, 4, 5,        6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,        23B, 23F, 24F, 33F and 35B.

The invention provides a multivalent immunogenic composition comprising22 distinct polysaccharide protein conjugates, wherein each of theconjugates comprises a capsular polysaccharide from a S. pneumoniaeserotype conjugated to a carrier protein, wherein the polysaccharide areprepared from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A,12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

The invention provides a multivalent immunogenic composition comprising22 distinct polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, and wherein the polysaccharide proteinconjugates include polysaccharides of a group of S. pneumoniae serotypesselected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A,12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

The invention provides a multivalent immunogenic composition comprising23 distinct polysaccharide protein conjugates, wherein each of theconjugates comprises a capsular polysaccharide from a S. pneumoniaeserotype conjugated to a carrier protein, wherein the polysaccharide areprepared from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

The invention provides a multivalent immunogenic composition comprising23 distinct S. pneumoniae polysaccharide protein conjugates, whereineach of the conjugates comprises a capsular polysaccharide from a S.pneumoniae serotype conjugated to a carrier protein, wherein eachdistinct polysaccharide protein conjugate comprises a polysaccharidefrom S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F,14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B,respectively, and wherein the carrier protein is CRM197.

The invention provides a multivalent immunogenic composition comprising24 distinct polysaccharide protein conjugates, wherein each of theconjugates comprises a capsular polysaccharide from a S. pneumoniaeserotype conjugated to a carrier protein, wherein the polysaccharide areprepared from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B.

The invention provides a multivalent immunogenic composition comprising24 distinct S. pneumoniae polysaccharide protein conjugates, whereineach of the conjugates comprises a capsular polysaccharide from a S.pneumoniae serotype conjugated to a carrier protein, wherein eachdistinct polysaccharide protein conjugate comprises a polysaccharidefrom S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B,respectively, and wherein the carrier protein is CRM197.

The invention provides a multivalent immunogenic composition comprising24 distinct polysaccharide protein conjugates, wherein each of theconjugates comprises a capsular polysaccharide from a S. pneumoniaeserotype conjugated to a carrier protein, wherein the polysaccharide areprepared from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B.

The invention provides a multivalent immunogenic composition comprising24 distinct S. pneumoniae polysaccharide protein conjugates, whereineach of the conjugates comprises a capsular polysaccharide from a S.pneumoniae serotype conjugated to a carrier protein, wherein eachdistinct polysaccharide protein conjugate comprises a polysaccharidefrom S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B,respectively, and wherein the carrier protein is CRM197.

The invention provides a multivalent immunogenic composition comprisingup to 33 distinct polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, and wherein the polysaccharide proteinconjugates include polysaccharides of a group of S. pneumoniae serotypesselected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B, further including one, two, three, four, five, six, seven, eight ornine additional S. pneumoniae serotypes selected from 7C, 9N, 16F, 21,23A, 31, 34, 35F and 38.

The invention provides a multivalent immunogenic composition comprisingup to 30 distinct polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, and wherein the polysaccharide proteinconjugates include polysaccharides of a group of S. pneumoniae serotypesselected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B, further including one, two, three, four, five or six additional S.pneumoniae serotypes selected from 7C, 9N, 16F, 23A, 35F and 38.

The invention provides a multivalent immunogenic composition comprisingup to 33 distinct polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, and wherein the polysaccharide proteinconjugates include polysaccharides of a group of S. pneumoniae serotypesselected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B, further including one, two, three, four, five, six, seven, eight ornine additional S. pneumoniae serotypes selected from 7C, 9N, 16F, 21,23A, 31, 34, 35F and 38.

The invention provides a multivalent immunogenic composition comprisingup to 30 distinct polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, and wherein the polysaccharide proteinconjugates include polysaccharides of a group of S. pneumoniae serotypesselected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B, further including one, two, three, four, five or six additional S.pneumoniae serotypes selected from 7C, 9N, 16F, 23A, 35F and 38.

In some embodiments, at least one of the polysaccharide proteinconjugates is formed by a conjugation reaction comprising an aproticsolvent, e.g. dimethylsulfoxide (DMSO).

In specific embodiments, each of the polysaccharide protein conjugatesis formed by a conjugation reaction comprising an aprotic solvent, e.g.(DMSO).

Also provided are methods for inducing a protective immune response in ahuman patient comprising administering the multivalent immunogeniccompositions of the invention to the patient. In some embodiments of themethods of the invention, the patient was previously treated with amultivalent pneumococcal vaccine.

A multivalent immunogenic composition of the invention may be used aspart of a treatment regimen with a different, complementary pneumococcalvaccine. Accordingly, the invention provides a method of inducing aprotective immune response in a human patient comprising administering amultivalent immunogenic composition of the invention to the patient,further comprising administering a multivalent pneumococcal vaccine tothe patient in any order. In particular embodiments, the multivalentpneumococcal vaccine is comprised of multiple S. pneumoniaepolysaccharide protein conjugates wherein each of the conjugatescomprises polysaccharide from an S. pneumoniae serotype conjugated to acarrier protein. In other embodiments, the multivalent pneumococcalvaccine is comprised of unconjugated capsular polysaccharides.

The invention also provides multivalent immunogenic compositionscomprising S. pneumoniae polysaccharide protein conjugates wherein eachof the conjugates comprises a polysaccharide from a S. pneumoniaeserotype conjugated to a carrier protein, wherein select serotypes of S.pneumoniae provide cross-reactivity to other select serotypes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Pre-immune (Pre), post-dose 1 (PD1), 2 (PD2) and 3 (PD3) IgGantibody dilution titers as determined by ECL for mice immunized withPCV22 unadjuvanted (PCV22 unadj) or formulated with aluminum phosphateadjuvant (PCV22/APA). Reading from left to right; Pre PCV22 unadj, PrePCV22/APA, PD1 PCV22 unadj, PD1 PCV22/APA, PD2 PCV22 unadj, PD2PCV22/APA, PD3 PCV22 unadj, and PD3 PCV22/APA.

FIG. 2. ECL dilution titer ratio of PCV22/APA compared to PCV22unadjuvanted (PCV22 unadj) at PD3.

FIG. 3. Serotype specific OPA dilution titers (pre-immune, PD1, PD2,PD3) for mice immunized with PCV22 unadjuvanted (PCV22 unadj) orformulated with APA (PCV22/APA). Reading from left to right; Pre PCV22unadj, Pre PCV22/APA, PD1 PCV22 unadj, PD1 PCV22/APA, PD2 PCV22 unadj,PD2 PCV22/APA, PD3 PCV22 unadj, and PD3 PCV22/APA.

FIG. 4. OPA dilution titer ratio of PCV22/APA compared to PCV22unadjuvanted (PCV22 unadj) at PD3.

FIG. 5. PCV22 immunized mice are protected from S. pneumoniae 24F intratracheal challenge.

FIG. 6. Pre-immune (Pre), PD1 and PD2 IgG antibody dilution titers asdetermined by ECL for rabbits immunized with PCV22 unadjuvanted orPCV22/APA. Error bars represent the 95% confidence intervals (CI) of thegeometric mean titer (GMT). Reading from left to right; Pre PCV22 unadj,Pre PCV22/APA, PD1 PCV22 unadj, PD1 PCV22/APA, PD2 PCV22 unadj, and PD2PCV22/APA.

FIG. 7. ECL GMT ratio of PCV22/APA compared to PCV22 unadjuvanted atPD2. Error bars represent the 95% confidence intervals (CI).

FIG. 8. Serotype specific OPA dilution titers (pre-immune “Pre” and PD2)for rabbits immunized with PCV22 unadjuvanted or PCV22/APA. Error barsrepresent the variation in functional antibody titers for five rabbits.

FIGS. 9A-9D. Pre-immune (Pre), PD1, PD2, and PD3 IgG antibody dilutiontiters as determined by ECL for IRMs immunized with FIG. 9A) PCV23unadjuvanted, FIG. 9B) PCV23 (DMSO)/APA, FIG. 9C) PCV23(DMSO+Aq)/APA,and FIG. 9D) PCV15/APA+PCV8/APA. Error bars represent the 95% confidenceintervals (CI) of the geometric mean titer (GMT).

FIGS. 10A-10C. Comparison of ECL antibody responses in IRMs (8-9 pergroup) following vaccination with PCV23 with or without APA. Symbolsindicate ratios at FIG. 10A) PD1, FIG. 10B) PD2, or FIG. 10C) PD3. GMTratios with error bars representing the 95% CIs.

FIGS. 11A-11C. Comparison of ECL antibody responses in IRMs (9 pergroup) following vaccination with PCV23 (DMSO+Aq)/APA orco-administrated with PCV15/APA+PCV8/APA. Symbols indicate ratios atFIG. 11A) PD1, FIG. 11B) PD2, and FIG. 11C) PD3. GMT ratios with errorbars representing the 95% CIs.

FIGS. 12A-12E. Comparison of boosted ECL antibody responses in IRMs (8-9per group) following vaccination with PCV23 unadjuvanted,PCV23(DMSO)/APA, PCV23(DMSO+Aq)/APA or PCV15/APA+PCV8/APA. FIG. 12A)PD1/Pre, FIG. 12B) PD2/Pre, FIG. 12C) PD3/Pre, FIG. 12D) PD2/PD1 andFIG. 12E) PD3/PD2. Symbols are GMT ratios with error bars representingthe 95% CIs.

FIGS. 13A-13B. (FIG. 13A) Pre-immune (Pre), post-dose 1 (PD1) and 2(PD2) IgG antibody dilution titers as determined by ECL for New Zealandwhite rabbits immunized with PCV24 formulated with aluminum phosphateadjuvant (PCV24/APA). Error bars represent the 95% confidence intervals(CI) of the geometric mean titer (GMT). (FIG. 13B) Serotype specific OPAdilution titers (pre-immune and PD2) for NZWRs immunized with PCV24/APA.Error bars represent the variation in functional antibody titers foreight NZWRs.

FIGS. 14A-14B. (FIG. 14A) Pre-immune (Pre), post-dose 1 (PD1), 2 (PD2)and 3 (PD3) IgG antibody dilution titers as determined by ECL for infantRhesus monkeys (IRMs) immunized with PCV24 formulated with aluminumphosphate adjuvant (PCV24/APA). Error bars represent the 95% confidenceintervals (CI) of the geometric mean titer (GMT). (FIG. 14B) Serotypespecific OPA dilution titers (pre-immune and PD3) for IRMs immunizedwith PCV24/APA. Error bars represent the variation in functionalantibody titers for five IRMs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides multivalent immunogenic compositionscomprising pneumococcal polysaccharide-protein conjugates, wherein eachof the conjugates comprises a polysaccharide from an S. pneumoniaeserotype conjugated to a carrier protein, wherein the serotypes of S.pneumoniae are as defined herein.

In some embodiments the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugates,wherein each of the conjugates comprises a polysaccharide from a S.pneumoniae serotype conjugated to a carrier protein, and wherein thepolysaccharide protein conjugates include polysaccharides of a group ofS. pneumoniae serotypes selected from the group consisting of:

-   -   a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A,        DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, DeOAc15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   cc) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   dd) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, DeOAc15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, DeOAc15B,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ll) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   nn) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   qq) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   rr) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   tt) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   uu) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ww) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   zz) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; and    -   bbb) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39.

In some embodiments, the multivalent immunogenic composition comprisespneumococcal serotypes selected from the group consisting of: i) 1, 3,4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F,14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or iii) 1,3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F and 35B; or iv) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B. In a particular embodiment, a multivalent immunogenic compositionof the invention comprises multiple pneumococcal S. pneumoniaepolysaccharide protein conjugates wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype conjugated toa carrier protein, wherein the serotypes of S. pneumoniae compriseserotypes: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C,19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5, 6A, 6B,7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F,33F and 35B; or iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14,15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. Saidcompositions were found to be immunogenic in mice, rabbits and/ormonkeys and generate functional antibody which killed vaccine-typebacterial strains at all doses tested.

The multivalent immunogenic compositions of the invention are useful forimmunizing a patient against vaccine-type S. pneumoniae serotypes and/oras part of a treatment regimen with different, complementarypneumococcal vaccine(s). Accordingly, the invention provides a method ofinducing a protective immune response in a human patient comprisingadministering a multivalent immunogenic composition of the invention tothe patient, and further comprising administering a multivalentpneumococcal vaccine to the patient, in any order. In other embodiments,the multivalent immunogenic compositions of the invention areadministered to a patient who had been previously immunized with adifferent multivalent pneumococcal vaccine.

In embodiments of the invention, conjugates from at least onepneumococcal serotype are prepared using reductive amination in anaprotic solvent such as DMSO. In further embodiments, the multivalentimmunogenic composition comprises pneumococcal conjugates that are eachprepared using reductive amination in an aprotic solvent. The use ofDMSO solvent enhances the covalent associations of polysaccharide toprotein through direct consumption of lysine residues on the surface ofthe carrier protein. The increased covalent association has a directbenefit to increasing the stability of the polysaccharide proteinconjugate of multivalent immunogenic compositions comprisingpolysaccharide antigens conjugated in DMSO.

I. Definitions and Abbreviations

As used throughout the specification and appended claims, the followingabbreviations apply:

APA aluminum phosphate adjuvant

-   -   APC antigen presenting cell    -   CI confidence interval    -   DMSO dimethylsulfoxide    -   DS polysaccharide-protein Drug Substance    -   GMC geometric mean concentration    -   GMT geometric mean titer    -   HPSEC high performance size exclusion chromatography    -   IM intra-muscular or intra-muscularly    -   IRM infant rhesus macaque    -   LOS lipo-oligosaccharide    -   LPS lipopolysaccharide    -   MALS multi-angle light scattering    -   MBC monovalent bulk conjugate    -   Mn number averaged molecular weight    -   MOPA multiplexed opsonophagocytic assays    -   MW molecular weight    -   NMWCO nominal molecular weight cut off    -   NZWR New Zealand White rabbit    -   OPA opsonophagocytosis assay    -   PCV pneumococcal conjugate vaccine    -   PD1 post-dose 1    -   PD2 post-dose 2    -   PD3 post-dose 3    -   PnPs Pneumococcal Polysaccharide    -   Ps polysaccharide    -   PS-20 polysorbate-20    -   RI refractive index    -   UV ultraviolet    -   w/v weight per volume

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used throughout the specification and in the appended claims, thesingular forms “a,” “an,” and “the” include the plural reference unlessthe context clearly dictates otherwise.

Reference to “or” indicates either or both possibilities unless thecontext clearly dictates one of the indicated possibilities. In somecases, “and/or” was employed to highlight either or both possibilities.

The terms “aqueous solvent” or “aqueous conditions” when used withconjugation, such as reductive amination, refers to use of water as thesolvent for the conjugation reaction. The water may contain buffers andother components except that no organic solvent is present.

The terms “aprotic solvent”, “DMSO solvent” or “DMSO conditions” whenused with conjugation, such as reductive amination, refers to use of anaprotic solvent, or a combination of aprotic solvents, (or DMSO, asapplicable) as the solvent for the conjugation reaction. The aproticsolvent may have some water present, for example, up to 1%, 2%, 5%, 10%or 20%.

The term “comprises” when used with the immunogenic composition of theinvention refers to the inclusion of any other components, such asadjuvants and excipients, or the addition of one or morepolysaccharide-protein conjugates that are not specifically enumerated.The term “consisting of” when used with the multivalentpolysaccharide-protein conjugate mixture refers to a mixture havingthose particular S. pneumoniae polysaccharide protein conjugates and noother S. pneumoniae polysaccharide protein conjugates from a differentserotype. “Consists essentially of” and variations such as “consistessentially of” or “consisting essentially of,” indicate the inclusionof any recited elements or group of elements, and the optional inclusionof other elements, of similar or different nature than the recitedelements, which do not materially change the basic or novel propertiesof the specified dosage regimen, method, or composition.

“Effective amount” of a composition of the invention refers to a doserequired to elicit antibodies that significantly reduce the likelihoodor severity of infectivity of a microbe, e.g., S. pneumoniae, during asubsequent challenge.

As used herein, the phrase “indicated for the prevention of pneumococcaldisease” means that a vaccine or immunogenic composition is approved byone or more regulatory authorities, such as the US Food and DrugAdministration, for the prophylaxis of one or more diseases caused byany serotype of S. pneumoniae, including, but not limited to:pneumococcal disease generally, pneumococcal pneumonia, pneumococcalmeningitis, pneumococcal bacteremia, invasive disease caused by S.pneumoniae, and otitis media caused by S. pneumoniae.

A “multivalent pneumococcal vaccine” is a pharmaceutical preparationcomprising more than one active agent (e.g., pneumococcal capsularpolysaccharide or pneumococcal polysaccharide protein conjugate) thatprovides active immunity to disease or pathological condition caused bymore than one serotype of S. pneumoniae.

The term “polysaccharide” is meant to include any antigenic saccharideelement (or antigenic unit) commonly used in the immunologic andbacterial vaccine arts, including, but not limited to, a “saccharide”,an “oligosaccharide”, a “polysaccharide”, a “liposaccharide”, a“lipo-oligosaccharide (LOS)”, a “lipopolysaccharide (LPS)”, a“glycosylate”, a “glycoconjugate” and the like.

The term “unadjuvanted”, in the context of a vaccine or immunogeniccomposition of the instant invention, means a pneumococcalpolysaccharide composition, including but not limited to PCV8, PCV15,PCV22, PCV23 and PCV24, wherein the composition contains no adjuvant.

“PCV8” refers to an immunogenic composition containing S. pneumoniaepolysaccharide (PnPs) serotypes -8, -10A, -12F, -15A, -15C, -23B, -24Fand -35B.

“PCV15” refers to an immunogenic composition containing S. pneumoniaepolysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -9V, -14,-18C, -19A, -19F, -22F, -23F, and -33F.

“PCV22” refers to an immunogenic composition containing S. pneumoniaepolysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -9V,-10A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F,-33F, and -35B.

“PCV23” refers to an immunogenic composition containing S. pneumoniaepolysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8, -9V,-10A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F,-33F, and -35B.

“PCV24” refers to an immunogenic composition containing S. pneumoniaepolysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8, -9V,-10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F,-24F, -33F, and -35B.

“CpG-containing nucleotide,” “CpG-containing oligonucleotide,” “CpGoligonucleotide,” and similar terms refer to a nucleotide molecule of6-50 nucleotides in length that contains an unmethylated CpG moiety.See, e.g., Wang et al., 2003, Vaccine 21:4297. CpG-containingoligonucleotides include modified oligonucleotides using any syntheticinternucleoside linkages, modified base and/or modified sugar.

An “adjuvant,” as defined herein, is a substance that serves to enhancethe immunogenicity of an immunogenic composition of the invention. Animmune adjuvant may enhance an immune response to an antigen that isweakly immunogenic when administered alone, e.g., inducing no or weakantibody titers or cell-mediated immune response, increase antibodytiters to the antigen, and/or lowers the dose of the antigen effectiveto achieve an immune response in the individual. Thus, adjuvants areoften given to boost the immune response and are well known to theskilled artisan.

A “patient” (alternatively referred to herein as a “subject”) refers toa mammal capable of being infected with a S. pneumoniae. In preferredembodiments, the patient is a human. A patient can be treatedprophylactically or therapeutically. Prophylactic treatment providessufficient protective immunity to reduce the likelihood or severity of apneumococcal infection or the effects thereof, e.g., pneumococcalpneumonia. Therapeutic treatment can be performed to reduce the severityor prevent recurrence of a S. pneumoniae infection or the clinicaleffects thereof. Prophylactic treatment can be performed using amultivalent immunogenic composition of the invention, as describedherein. The composition of the invention can be administered to thegeneral population or to those persons at an increased risk ofpneumococcal infection, e.g. the elderly, or those who live with or carefor the elderly.

Those “in need of treatment” include those previously exposed to orinfected with S. pneumoniae, those who were previously vaccinatedagainst S. pneumoniae, as well as those prone to have an infection orany person in which a reduction in the likelihood of infection isdesired, e.g., the immunocompromised, the elderly, children, adults, orhealthy individuals.

A “stable” multivalent immunogenic composition is a composition whichhas no significant changes observed at a refrigerated temperature (e.g.,2-8° C. or 4° C.) for at least 1 month, 2 months, 3 months, 6 months, 12months and/or 24 months. Additionally, a “stable” composition includesone that exhibits desired features at temperatures including at 25° C.and 37° C. for periods including 1 month, 3 months, 6 months, 12 months,and/or 24 months. Typical acceptable criteria for stability are asfollows: no more than about 5%, about 10%, about 15%, or about 20%variability in one or more of the following: (a) the number averagemolecular weight (Mn) of the S. pneumoniae polysaccharide proteinconjugates in the composition, (b) weight average molecular weight (Mw)of the S. pneumoniae polysaccharide protein conjugates in thecomposition, (c) total polysaccharide concentration in the composition,(d) emission maximum of the composition measured using intrinsic proteinfluorescence spectroscopy at a particular excitation wavelength, e.g.280 nanometers, and (e) the fluorescence intensity of the compositionmeasured using intrinsic protein fluorescence spectroscopy at aparticular excitation wavelength. The term “stable” may also be used torefer to a particular pneumococcal conjugate within a multivalentimmunogenic composition. In such use, the term refers to a conjugatethat exhibits the desired properties over time, at a particulartemperature, and such properties vary no more that about 5%, about 10%,about 15%, or about 20% over the time and temperature noted.

II. Multivalent Immunogenic Compositions

The invention provides multivalent immunogenic compositions comprisingmultiple S. pneumoniae polysaccharide protein conjugates wherein each ofthe conjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein. Different aspects and embodiments ofthe multivalent immunogenic compositions of the invention are described,infra.

In one embodiment (Embodiment E1), the invention provides a multivalentimmunogenic composition comprising multiple S. pneumoniae polysaccharideprotein conjugates, each comprising capsular polysaccharide from an S.pneumoniae serotype conjugated to a carrier protein, wherein theserotypes of S. pneumoniae comprise, consist, or consist essentially of:i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F and 35B or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35Bor iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In sub-embodiments ofEmbodiment E1, the immunogenic composition does not comprise any furtherS. pneumoniae polysaccharide protein conjugates.

As used herein, de-O-acetylated serotype 15B (DeOAc15B) pneumococcalpolysaccharide is substantially equivalent to serotype 15C pneumococcalpolysaccharide and has a substantially identical NMR spectra (data notshown). As used herein, de-O-acetylated serotype 15B pneumococcalpolysaccharide and serotype 15C pneumococcal polysaccharide may eachhave an O-Acetyl content per repeating unit in the range of 0-5%, or inthe range of 0-4%, or in the range of 0-3%, or in the range of 0-2%, orin the range of 0-1%, or in the range of 0-0.5%, or in the range of0-0.1%, or no O-acetyl content. In a report by Spencer B. L., et al.,pneumococcal polysaccharide 15C may be slightly O-acetylated (Spencer,B. L. et al., Clin. Vac. Immuno. (2017) 24(8): 1-13). Thus, in any ofthe embodiments of the multivalent immunogenic compositions herein,de-O-acetylated serotype 15B (DeOAc15B) can be used in place of serotype15C. Processes for de-O-acetylation are known in the art, for example asdescribed in Rajam et al., Clinical and Vaccine Immunology, 2007, 14(9):1223-1227.

In certain embodiments of any of the multivalent immunogeniccompositions of the invention, including Embodiment E1 and anysub-embodiment thereof, the composition further comprises apharmaceutically acceptable carrier.

Cross-Reactivity

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from a S.pneumoniae serotype, including serotype 6C, conjugated to a carrierprotein, wherein serotype 6C of S. pneumoniae provides cross-reactivityagainst serotypes 6A and 6B of S. pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 6A, conjugated to a carrierprotein, wherein serotype 6A of S. pneumoniae provides cross-protectionagainst serotypes 6B and/or 6C of S. pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 6B, conjugated to a carrierprotein, wherein serotype 6B of S. pneumoniae provides cross-protectionagainst serotypes 6A and/or 6C of S. pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 15C, conjugated to a carrierprotein, wherein serotype 15C of S. pneumoniae provides cross-protectionagainst serotype 15B of S. pneumoniae.

In an embodiment the invention provides multivalent immunogeniccompositions comprising S. pneumoniae polysaccharide protein conjugateswherein each of the conjugates comprises a polysaccharide from an S.pneumoniae serotype, including serotype 15B, conjugated to a carrierprotein, wherein serotype 15B of S. pneumoniae provides cross-protectionagainst serotype 15C of S. pneumoniae.

Carrier Protein

In particular embodiments of the present invention, CRM197 is used asthe carrier protein. CRM197 is a non-toxic variant (i.e., toxoid) ofdiphtheria toxin having the following sequence of amino acids:

(SEQ ID NO: 1) GADDVVDSSK SFVMENFSSY HGTKPGYVDS IQKGIQKPKSGTQGNYDDDW KEFYSTDNKY DAAGYSVDNE NPLSGKAGGVVKVTYPGLTK VLALKVDNAE TIKKELGLSL TEPLMEQVGTEEFIKRFGDG ASRVVLSLPF AEGSSSVEYI NNWEQAKALSVELEINFETR GKRGQDAMYE YMAQACAGNR VRRSVGSSLSCINLDWDVIR DKTKTKIESL KEHGPIKNKM SESPNKTVSEEKAKQYLEEF HQTALEHPEL SELKTVTGTN PVFAGANYAAWAVNVAQVID SETADNLEKT TAALSILPGI GSVMGIADGAVHHNTEEIVA QSIALSSLMV AQAIPLVGEL VDIGFAAYNFVESIINLFQV VHNSYNRPAY SPGHKTQPFL HDGYAVSWNTVEDSIIRTGF QGESGHDIKI TAENTPLPIA GVLLPTIPGKLDVNKSKTHI SVNGRKIRMR CRAIDGDVTF CRPKSPVYVGNGVHANLHVA FHRSSSEKIH SNEISSDSIG VLGYQKTVDH TKVNSKLSLF FEIKS

In one embodiment, CRM197 is isolated from cultures of Corynebacteriumdiphtheria strain C7 (β197) grown in casamino acids and yeastextract-based medium. In another embodiment, CRM197 is preparedrecombinantly in accordance with the methods described in U.S. Pat. No.5,614,382. Typically, CRM197 is purified through a combination ofultra-filtration, ammonium sulfate precipitation, and ion-exchangechromatography. In some embodiments, CRM197 is prepared in Pseudomonasfluorescens using Pfenex Expression Technology™ (Pfenex Inc., San Diego,Calif.).

Other suitable carrier proteins include additional inactivated bacterialtoxins such as DT (Diphtheria toxoid) or fragment B of DT (DTFB), TT(tetanus toxid) or fragment C of TT, pertussis toxoid, cholera toxoid(e.g., as described in WO 2004/083251), E. coli LT, E. coli ST, andexotoxin A from Pseudomonas aeruginosa. Bacterial outer membraneproteins such as outer membrane protein complex (OMPC), porins,transferrin binding proteins, pneumococcal surface protein A (PspA; SeeWO 02/091998), pneumococcal adhesin protein (PsaA), C5a peptidase fromGroup A or Group B streptococcus, or Haemophilus influenzae protein D,pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63; 2706-13)including ply detoxified in some fashion for example dPLY-GMBS (See WO04/081515) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE andfusions of Pht proteins for example PhtDE fusions, PhtBE fusions (See WO01/98334 and WO 03/54007), can also be used. Other proteins, such asovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA)or purified protein derivative of tuberculin (PPD), PorB (from N.meningitidis), PD (Haemophilus influenzae protein D; see, e.g., EP 0 594610 B), or immunologically functional equivalents thereof, syntheticpeptides (See EP0378881 and EP0427347), heat shock proteins (See WO93/17712 and WO 94/03208), pertussis proteins (See WO 98/58668 andEP0471177), cytokines, lymphokines, growth factors or hormones (See WO91/01146), artificial proteins comprising multiple human CD4+ T cellepitopes from various pathogen derived antigens (See Falugi et al.,2001, Eur J Immunol 31:3816-3824) such as N19 protein (See Baraldoi etal., 2004, Infect Immun 72:4884-7), iron uptake proteins (See WO01/72337), toxin A or B of C. difficile (See WO 00/61761), and flagellin(See Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used ascarrier proteins.

Other DT mutants can be used as the carrier protein, such as CRM176,CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218:3838-3844); CRM9,CRM45, CRM102, CRM103 and CRM107 and other mutations described byNicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, MaecelDekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Serand/or Ala 158 to Gly and other mutations disclosed in U.S. Pat. No.4,709,017 or 4,950,740; mutation of at least one or more residues Lys516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed inU.S. Pat. No. 5,917,017 or 6,455,673; or fragment disclosed in U.S. Pat.No. 5,843,711. Such DT mutants can also be used to make DTFB variantswhere the variants comprise the B fragment contain the epitope regions.

In certain embodiments, the carrier protein is selected from the groupconsisting of: Outer Membrane Protein Complex (OMPC), tetanus toxoid,diphtheria toxoid, protein D and CRM197.

In some embodiments of the invention, a second carrier can be used forone or more of the polysaccharide protein conjugates in the multivalentimmunogenic composition. The second carrier protein is preferably aprotein that is non-toxic and non-reactogenic and obtainable insufficient amount and purity. The second carrier protein is alsoconjugated or joined with the S. pneumoniae polysaccharide to enhanceimmunogenicity of the antigen. Carrier proteins should be amenable tostandard conjugation procedures. In one embodiment, each capsularpolysaccharide not conjugated to the first carrier protein is conjugatedto the same second carrier protein (e.g., each capsular polysaccharidemolecule being conjugated to a single carrier protein). In anotherembodiment, the capsular polysaccharides not conjugated to the firstcarrier protein are conjugated to two or more carrier proteins (eachcapsular polysaccharide molecule being conjugated to a single carrierprotein). In such embodiments, each capsular polysaccharide of the sameserotype is typically conjugated to the same carrier protein.

In embodiments of the invention, including Embodiment E1 and anysub-embodiment thereof, one or more (including 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more, where applicable) of the polysaccharide serotypes isconjugated to CRM197. In further embodiments of the invention, includingEmbodiment E1 and any sub-embodiment thereof, each of the polysaccharideserotypes is conjugated to CRM197.

Formulation of the polysaccharide-protein conjugates of the presentinvention can be accomplished using art-recognized methods. Forinstance, individual pneumococcal conjugates can be formulated with aphysiologically acceptable vehicle to prepare the composition. Examplesof such vehicles include, but are not limited to, water, bufferedsaline, polyols (e.g., glycerol, propylene glycol, liquid polyethyleneglycol) and dextrose solutions.

In a preferred embodiment, the vaccine composition is formulated inL-histidine buffer with sodium chloride.

In some embodiments of the invention, the multivalent immunogeniccomposition comprises multiple S. pneumoniae polysaccharide proteinconjugates comprising capsular polysaccharide from an S. pneumoniaeserotype conjugated to a carrier protein and an adjuvant, wherein the S.pneumoniae serotypes are as described herein. Suitable adjuvants toenhance effectiveness of the composition include, but are not limitedto:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example, (a) MF59(International Patent Application Publication No. WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, (c) Ribi™ adjuvant system (RAS), (Corixa,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting of3-O-deacylated monophosphorylipid A (MPL™) described in U.S. Pat. No.4,912,094, trehalose dimycolate (TDM), and cell wall skeleton (CWS),preferably MPL+CWS (Detox™); and (d) a Montanide ISA;

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, Mass.) (see, e.g., U.S. Pat. No. 5,057,540) may be used orparticles generated therefrom such as ISCOM (immunostimulating complexesformed by the combination of cholesterol, saponin, phospholipid, andamphipathic proteins) and Iscomatrix® (having essentially the samestructure as an ISCOM but without the protein);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-β-D-glucopyranoside,which is also known as 529 (formerly known as RC529), which isformulated as an aqueous form or as a stable emulsion

(5) synthetic polynucleotides such as oligonucleotides containing CpGmotif(s) (U.S. Pat. No. 6,207,646);

(6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF),costimulatory molecules B7-1 and B7-2, etc; and

(7) complement, such as a trimer of complement component C3d.

In another embodiment, the adjuvant is a mixture of 2, 3, or more of theabove adjuvants, e.g., SBAS2 (an oil-in-water emulsion also containing3-deacylated monophosphoryl lipid A and QS21).

Muramyl peptides include, but are not limited to,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′, 2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In certain embodiments, the adjuvant is an aluminum salt. The aluminumsalt adjuvant may be an alum-precipitated vaccine or an alum-adsorbedvaccine. Aluminum-salt adjuvants are well known in the art and aredescribed, for example, in Harlow, E. and D. Lane (1988; Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992;Aluminum salts. Research in Immunology 143:489-493). The aluminum saltincludes, but is not limited to, hydrated alumina, alumina hydrate,alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate,alhydrogel, Superfos, Amphogel, aluminum (III) hydroxide, aluminumhydroxyphosphate sulfate, Aluminum Phosphate Adjuvant (APA), amorphousalumina, trihydrated alumina, or trihydroxyaluminum.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA ismanufactured by blending aluminum chloride and sodium phosphate in a 1:1volumetric ratio to precipitate aluminum hydroxyphosphate. After theblending process, the material is size-reduced with a high-shear mixerto achieve a monodisperse particle size distribution. The product isthen diafiltered against physiological saline and steam sterilized. Inone embodiment, the dose of the aluminum salt is 10, 15, 20, 25, 30, 50,70, 100, 125, 150, 200, 300, 500, or 700 gg, or 1, 1.2, 1.5, 2, 3, 5 mgor more. In yet another embodiment, the dose of alum salt describedabove is per μg of recombinant protein.

In certain embodiments, a commercially available Al(OH)₃ (e.g.Alhydrogel or Superfos of Denmark/Accurate Chemical and Scientific Co.,Westbury, N.Y.) is used to adsorb proteins in a ratio of 50-200 μgprotein/mg aluminum hydroxide. Adsorption of protein is dependent, inanother embodiment, on the pI (Isoelectric pH) of the protein and the pHof the medium. A protein with a lower pI adsorbs to the positivelycharged aluminum ion more strongly than a protein with a higher pI.Aluminum salts may establish a depot of antigen that is released slowlyover a period of 2-3 weeks, be involved in nonspecific activation ofmacrophages and complement activation, and/or stimulate innate immunemechanism (possibly through stimulation of uric acid). See, e.g.,Lambrecht et al., 2009, Curr Opin Immunol 21:23.

Monovalent bulk aqueous conjugates are typically blended together anddiluted to target 4 μg/mL for all serotypes except 6B, which may bediluted to target 8 μg/mL. Once diluted, the batch will be filtersterilized, and an equal volume of aluminum phosphate adjuvant addedaseptically to target a final aluminum concentration of 250 μg/mL. Theadjuvanted, formulated batch will be filled into single-use, 0.5 mL/dosevials.

In certain embodiments, the adjuvant is a CpG-containing nucleotidesequence, for example, a CpG-containing oligonucleotide, in particular,a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment,the adjuvant is ODN 1826, which may be acquired from ColeyPharmaceutical Group.

Methods for use of CpG oligonucleotides are well known in the art andare described, for example, in Sur et al., 1999, J Immunol. 162:6284-93;Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006,Crit Rev Ther Drug Carrier Syst. 23:89-110.

In alternative embodiments, the immunogenic composition comprisesmultiple S. pneumoniae polysaccharide protein conjugates as describedherein, for example in Embodiment E1 or any sub-embodiment thereof, anddoes not comprise an adjuvant.

Formulations

The multivalent immunogenic compositions of the invention can beformulated as single dose vials, multi-dose vials or as pre-filled glassor plastic syringes.

In another embodiment, the multivalent immunogenic compositions of thepresent invention are administered orally, and are thus formulated in aform suitable for oral administration, i.e., as a solid or a liquidpreparation. Solid oral formulations include tablets, capsules, pills,granules, pellets and the like. Liquid oral formulations includesolutions, suspensions, dispersions, emulsions, oils and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueousor nonaqueous solutions, suspensions, emulsions or oils. Examples ofnonaqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Examples of oils are those of animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipidfrom milk or eggs.

The multivalent immunogenic compositions of the instant invention may beisotonic, hypotonic or hypertonic. However, it is often preferred that acomposition for infusion or injection be essentially isotonic, whenadministrated. Hence, for storage, a composition may preferably beisotonic or hypertonic. If the composition is hypertonic for storage, itmay be diluted to become an isotonic solution prior to administration.

The isotonic agent may be an ionic isotonic agent such as a salt or anon-ionic isotonic agent such as a carbohydrate. Examples of ionicisotonic agents include but are not limited to NaCl, CaCl₂, KCl andMgCl₂. Examples of non-ionic isotonic agents include but are not limitedto mannitol, sorbitol and glycerol.

It is also preferred that at least one pharmaceutically acceptableadditive is a buffer. For some purposes, for example, when thepharmaceutical composition is meant for infusion or injection, it isoften desirable that the composition comprises a buffer, which iscapable of buffering a solution to a pH in the range of 4 to 10, such as5 to 9, for example 6 to 8.

The buffer may, for example, be selected from the group consisting ofTRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate,citrate, carbonate, glycinate, histidine, glycine, succinate andtriethanolamine buffer.

The buffer may be selected from USP compatible buffers for parenteraluse, in particular, when the pharmaceutical formulation is forparenteral use. For example the buffer may be selected from the groupconsisting of monobasic acids such as acetic, benzoic, gluconic,glyceric and lactic; dibasic acids such as aconitic, adipic, ascorbic,carbonic, glutamic, malic, succinic and tartaric, polybasic acids suchas citric and phosphoric; and bases such as ammonia, diethanolamine,glycine, triethanolamine, and TRIS.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, glycols such as propylene glycols orpolyethylene glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), andPoloxamer 188 (P188) are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, olive oil,sunflower oil, fish-liver oil, another marine oil, or a lipid from milkor eggs.

The formulations of the invention may also contain a surfactant.Preferred surfactants include, but are not limited to: Poloxamer -188(P188; Pluoronic; F68 NF), the polyoxyethylene sorbitan esterssurfactants (commonly referred to as the Tweens), especially PS-20 andPS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/orbutylene oxide (BO), sold under the DOWFAX™ tradename, such as linearEO/PO block copolymers; octoxynols, which can vary in the number ofrepeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (TritonX-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. A preferred surfactant for including inthe emulsion is PS-80.

Mixtures of surfactants can be used, e.g. PS-80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (PS-80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as PS-80) of from 0.01 to 1%, in particular about0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, orother detergents in the Triton series) of from 0.001 to 0.1%, inparticular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) offrom 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% orabout 0.5%.

In certain embodiments, the composition consists essentially ofhistidine (20 mM), saline (150 mM) and 0.2% PS-20 at a pH of 5.8 with250 μg/mL of APA (Aluminum Phosphate Adjuvant). PS-20 can range from0.005% to 0.3% (w/v). In another embodiment, PS-20 can range from 0.025%to 0.8% (w/v). In another embodiment, PS-20 can range from 0.05% to 0.8%(w/v). In another embodiment, PS-20 can range from 0.05% to 0.2% (w/v).The process consists of combining a blend of up to 24 serotypes inhistidine, saline, and PS-20, then combining this blended material withAPA and saline with or without antimicrobial preservatives.

In particular embodiments, the multivalent immunogenic compositioncomprises S. pneumoniae polysaccharide protein conjugates wherein eachof the conjugates comprises a polysaccharide from an S. pneumoniaeserotype conjugated to a carrier protein, wherein the serotypes of S.pneumoniae in the polysaccharide protein conjugates comprise any of thesets of serotypes set forth herein, and further comprises 20-80 mMhistidine pH 5.8 and 150 mM NaCl. In some embodiments, the multivalentimmunogenic composition further comprises from 0.2% to 0.8% w/vpolysorbate 20.

The multivalent immunogenic composition PCV24 is prepared byindividually conjugating the CRM197 protein to S. pneumoniaepolysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8, -9V,-10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F,-24F, -33F, and -35B using reductive amination in an aprotic solvent(also referred to as DMSO chemistry) and formulated in 20 mM L-HistidinepH 5.8, 150 mM NaCl and 0.1% w/v Polysorbate-20 (PS-20) at 4 μg/mL or 8μg/mL of each polysacchardide serotype for a total polysaccharideconcentration of 96 μg/mL or 192 μg/mL, respectively, and referred to as“PCV24 unadj”. In another specific embodiment, the multivalentimmunogenic composition PCV24 is prepared in 20 mM L-Histidine pH 5.8,150 mM NaCl and 0.2% w/v Polysorbate-20 (PS-20) at 4 μg/mL of eachpolysaccharide serotype for a total polysaccharide concentration of 96μg/mL further comprising 250 μg [Al]/mL in the form of AluminumPhosphate Adjuvant. This is referred to as “PCV24/APA”.

The choice of surfactant may need to be optimized for different drugproducts and drug substances. For multivalent vaccines having 15 or moreserotypes, PS-20 and P188 are preferred. The choice of chemistry used tomake conjugates can also play an important role in the stabilization ofthe formulation. In particular, when the conjugation reactions used toprepare different polysaccharide protein conjugates in a multivalentcomposition include both aqueous solvent and DMSO solvent, particularsurfactant systems provide significant differences in stability.Improved stability of polysacharide protein conjugates was seen withpolysorbate 20 alone or with poloxamer 188 in combination with a polyol.

The exact mechanism of how a specific detergent protects abiotherapeutic is poorly understood and cannot be predicted a priori.Possible stabilization mechanisms include preferential hydration,preferential exclusion, air/liquid interface competition betweenbiotherapeutic and surface, surface tension, and/or direct associationof the detergent with the biotherapeutic to mask hydrophobic patcheswhich serve as seeds for aggregation.

Poloxamer may also be used in the compositions of the invention. Apoloxamer is a nonionic triblock copolymer composed of a centralhydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked bytwo hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).Poloxamers are also known by the tradename Pluronic®. Because thelengths of the polymer blocks can be customized, many differentpoloxamers exist that have slightly different properties. For thegeneric term “poloxamer”, these copolymers are commonly named with theletter “P” (for poloxamer) followed by three digits, the first twodigits×100 give the approximate molecular mass of the polyoxypropylenecore, and the last digit×10 gives the percentage polyoxyethylene content(e.g., P407=Poloxamer with a polyoxypropylene molecular mass of 4,000g/mol and a 70% polyoxyethylene content). For the Pluronic® tradename,coding of these copolymers starts with a letter to define its physicalform at room temperature (L=liquid, P=paste, F=flake (solid)) followedby two or three digits. The first digit (two digits in a three-digitnumber) in the numerical designation, multiplied by 300, indicates theapproximate molecular weight of the hydrophobe; and the last digit×10gives the percentage polyoxyethylene content (e.g., L61=Pluronic® with apolyoxypropylene molecular mass of 1,800 g/mol and a 10% polyoxyethylenecontent). See U.S. Pat. No. 3,740,421.

Examples of poloxamers have the general formula:HO(C₂H₄O)(C₃H₆O)_(b)(C₂HO)_(a)H, wherein a and b blocks have thefollowing values:

Pluronic ® Poloxamer A B Molecular Weight L31 2 16 1100 (average) L351900 (average) L44NF 124 12 20 2090 to 2360 L64 2900 (average) L81 2800(average) L121 4400 (average) P123 20 70 5750 (average) F68NF 188 80 277680 to 9510 F87NF 237 64 37 6840 to 8830 F108NF 338 141 44 12700 to17400 F127NF 407 101 56  9840 to 14600

Molecular weight units, as used herein, are in Dalton (Da) or g/mol.

Preferably, the poloxamer generally has a molecular weight in the rangefrom 1,100 to 17,400 Da, from 7,500 to 15,000 Da, or from 7,500 to10,000 Da. The poloxamer can be selected from poloxamer 188 or poloxamer407. The final concentration of the poloxamer in the formulations isfrom 0.001% to 5% weight/volume, or 0.025% to 1% weight/volume. Incertain aspects, the polyol is propylene glycol and is at finalconcentration from 1% to 20% weight/volume. In certain aspects, thepolyol is polyethylene glycol 400 and is at final concentration from 1%to 20% weight/volume.

Suitable polyols for the formulations of the invention are polymericpolyols, particularly polyether diols including, but are not limited to,propylene glycol and polyethylene glycol, Polyethylene glycol monomethylethers. Propylene glycol is available in a range of molecular weights ofthe monomer from ˜425 to -2,700. Polyethylene glycol and Polyethyleneglycol monomethyl ether is also available in a range of molecularweights ranging from ˜200 to ˜35,000 including but not limited toPEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000,PEG MME 3350 and PEG MME 4000. A preferred polyethylene glycol ispolyethylene glycol 400. The final concentration of the polyol in theformulations of the invention may be 1% to 20% weight/volume or 6% to20% weight/volume.

The formulation also contains a pH-buffered saline solution. The buffermay, for example, be selected from the group consisting of TRIS,acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate,carbonate, glycinate, histidine, glycine, succinate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. Thebuffer is capable of buffering a solution to a pH in the range of 4 to10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspect of the invention, thebuffer is selected from the group consisting of phosphate, succinate,histidine, MES, MOPS, HEPES, acetate or citrate. The buffer mayfurthermore, for example, be selected from USP compatible buffers forparenteral use, in particular, when the pharmaceutical formulation isfor parenteral use. In one embodiment, the concentration of buffer willrange from 1 mM to 100 mM. In another embodiment, the concentration ofbuffer will range from 10 mM to 80 mM. In another embodiment, theconcentration of buffer will range from 1 mM to 50 mM, or 5 mM to 50 mM.In certain aspects, the buffer is histidine at a final concentration of5 mM to 50 mM, or succinate at a final concentration of 1 mM to 10 mM.In certain aspects, the histidine buffer is at a final concentration of20 mM±2 mM.

While the saline solution (e.g., a solution containing NaCl) ispreferred, other salts suitable for formulation include but are notlimited to, CaCl₂), KCl and MgCl₂ and combinations thereof. Non-ionicisotonic agents including but not limited to sucrose, trehalose,mannitol, sorbitol and glycerol may be used in lieu of a salt. Suitablesalt ranges include, but are not limited to 20 mM to 500 mM or 40 mM to170 mM. In one aspect, the saline is NaCl, optionally present at aconcentration from 25 mM to 170 mM.

In a preferred embodiment, the formulations comprise a L-histidinebuffer with sodium chloride.

In another embodiment, the pharmaceutical composition is delivered in acontrolled release system. For example, the agent can be administeredusing intravenous infusion, a transdermal patch, liposomes, or othermodes of administration. In another embodiment, polymeric materials areused; e.g. in microspheres in or an implant.

The amount of conjugate in each dose of the composition is selected asan amount that induces an immunoprotective response without significant,adverse effects. Such amount can vary depending upon the pneumococcalserotype. Generally, for polysaccharide-based conjugates, each dose willcomprise 0.08 to 100 μg of each polysaccharide. In some embodiments ofthe invention, the dose of each polysaccharide conjugate is from 0.08 to10 μg. In further embodiments, the dose of each conjugate is from 1 to 5μg, from 0.4 to 4 μg, from 0.4 to 3 μg, from 0.4 to 2 μg, or from 0.4 to1 μg. In some embodiments, the dose of one or more polysaccharideconjugates is 100, 150, 200, 250, 300, 400, 500, or 750 ng or 0.4, 0.5,0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11,12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90, or 100μg.

In some embodiments of the compositions of the invention, all of thepolysaccharide conjugates are present in the composition in the sameamount. In further embodiments, the polysaccharide conjugates arepresent in the composition in different amounts (i.e., at least onepolysaccharide conjugate is present in an amount that is different thanone or more of the other polysaccharide conjugates of the composition).

Optimal amounts of components for a particular immunogenic compositioncan be ascertained by standard studies involving observation ofappropriate immune responses in subjects. For example, in anotherembodiment, the dosage for human vaccination is determined byextrapolation from animal studies to human data. In another embodiment,the dosage is determined empirically.

The compositions of this invention may also include one or more proteinsfrom S. pneumoniae. Examples of S. pneumoniae proteins suitable forinclusion include those identified in International Patent ApplicationPublication Nos. WO 02/083855 and WO 02/053761.

In certain embodiments, the compositions of the invention areadministered to a subject by one or more methods known to a personskilled in the art, such as parenterally, transmucosally, transdermally,intramuscularly, intravenously, intra-dermally, intra-nasally,subcutaneously, intra-peritonealy, and formulated accordingly. In oneembodiment, compositions of the present invention are administered viaepidermal injection, intramuscular injection, intravenous,intra-arterial, subcutaneous injection, or intra-respiratory mucosalinjection of a liquid preparation. Liquid formulations for injectioninclude solutions and the like.

III. Methods of Making

Capsular polysaccharides from Streptococcus pneumoniae can be preparedby standard techniques known to those skilled in the art. For example,polysaccharides can be isolated from bacteria and may be sized to somedegree by known methods (see, e.g., European Patent Nos. EP497524 andEP497525); and preferably by microfluidisation accomplished using ahomogenizer or by chemical hydrolysis. In one embodiment, eachpneumococcal polysaccharide serotype is grown in a soy-based medium. Theindividual polysaccharides are then purified through standard stepsincluding centrifugation, precipitation, and ultra-filtration. See,e.g., U.S. Patent Application Publication No. 2008/0286838 and U.S. Pat.No. 5,847,112. Polysaccharides can be sized in order to reduce viscosityin polysaccharide samples and/or to improve filterability for conjugatedproducts using techniques such as mechanical or chemical sizing.Chemical hydrolysis may be conducted using acetic acid. Mechanicalsizing may be conducted using High Pressure Homogenization Shearing.

The purified polysaccharides can be chemically activated to make thesaccharides capable of reacting with the carrier protein. The purifiedpolysaccharides can be connected to a linker. Once activated orconnected to a linker, each capsular polysaccharide is separatelyconjugated to a carrier protein to form a glycoconjugate. Thepolysaccharide conjugates may be prepared by known coupling techniques.

The polysaccharide can be coupled to a linker to form apolysaccharide-linker intermediate in which the free terminus of thelinker is an ester group. The linker is therefore one in which at leastone terminus is an ester group. The other terminus is selected so thatit can react with the polysaccharide to form the polysaccharide-linkerintermediate.

The polysaccharide can be coupled to a linker using a primary aminegroup in the polysaccharide. In this case, the linker typically has anester group at both termini. This allows the coupling to take place byreacting one of the ester groups with the primary amine group in thepolysaccharide by nucleophilic acyl substitution. The reaction resultsin a polysaccharide-linker intermediate in which the polysaccharide iscoupled to the linker via an amide linkage. The linker is therefore abifunctional linker that provides a first ester group for reacting withthe primary amine group in the polysaccharide and a second ester groupfor reacting with the primary amine group in the carrier molecule. Atypical linker is adipic acid N-hydroxysuccinimide diester (SIDEA).

The coupling can also take place indirectly, i.e. with an additionallinker that is used to derivatise the polysaccharide prior to couplingto the linker.

The polysaccharide is coupled to the additional linker using a carbonylgroup at the reducing terminus of the polysaccharide. This couplingcomprises two steps: (a1) reacting the carbonyl group with theadditional linker; and (a2) reacting the free terminus of the additionallinker with the linker. In these embodiments, the additional linkertypically has a primary amine group at both termini, thereby allowingstep (a1) to take place by reacting one of the primary amine groups withthe carbonyl group in the polysaccharide by reductive amination. Aprimary amine group is used that is reactive with the carbonyl group inthe polysaccharide. Hydrazide or hydroxylamino groups are suitable. Thesame primary amine group is typically present at both termini of theadditional linker. The reaction results in a polysaccharide-additionallinker intermediate in which the polysaccharide is coupled to theadditional linker via a C—N linkage.

The polysaccharide can be coupled to the additional linker using adifferent group in the polysaccharide, particularly a carboxyl group.This coupling comprises two steps: (a1) reacting the group with theadditional linker; and (a2) reacting the free terminus of the additionallinker with the linker. In this case, the additional linker typicallyhas a primary amine group at both termini, thereby allowing step (a1) totake place by reacting one of the primary amine groups with the carboxylgroup in the polysaccharide by EDAC activation. A primary amine group isused that is reactive with the EDAC-activated carboxyl group in thepolysaccharide. A hydrazide group is suitable. The same primary aminegroup is typically present at both termini of the additional linker. Thereaction results in a polysaccharide-additional linker intermediate inwhich the polysaccharide is coupled to the additional linker via anamide linkage.

In one embodiment, the chemical activation of the polysaccharides andsubsequent conjugation to the carrier protein by reductive amination canbe achieved by means described in U.S. Pat. Nos. 4,365,170, 4,673,574and 4,902,506, U.S. Patent Application Publication Nos. 2006/0228380,2007/184072, 2007/0231340 and 2007/0184071, and WO2006/110381,WO2008/079653, and WO2008/143709. The chemistry may include theactivation of pneumococcal polysaccharide by reaction with any oxidizingagent which oxidizes a terminal hydroxyl group to an aldehyde, such asperiodate (including sodium periodate, potassium periodate, or periodicacid). The reaction leads to a random oxidative cleavage of vicinalhydroxyl groups of the carbohydrates with the formation of reactivealdehyde groups.

Coupling to the carrier protein is by reductive amination via directamination to the lysyl groups of the protein. For example, conjugationcan be carried out by reacting a mixture of the activated polysaccharideand carrier protein with a reducing agent such as sodiumcyanoborohydride. The conjugation reaction may take place under aqueoussolution or in the presence of DMSO. See, e.g., US2015/0231270,US2011/0195086 and EP 0471 177 B1. Unreacted aldehydes are then cappedwith the addition of a strong reducing agent, such as sodiumborohydride.

Reductive amination involves two steps, (1) oxidation of thepolysaccharide to form reactive aldehydes, (2) reduction of the imine(Schiff base) formed between activated polysaccharide and a carrierprotein to form a stable amine conjugate bond. Before oxidation, thepolysaccharide is optionally size reduced. Mechanical methods (e.g.homogenization) or chemical hydrolysis may be employed. Chemicalhydrolysis may be conducted using acetic acid. The oxidation step mayinvolve reaction with periodate. For the purpose of the presentinvention, the term “periodate” includes both periodate and periodicacid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁻) and includes the various salts of periodate(e.g., sodium periodate and potassium periodate). In an embodiment thecapsular polysaccharide is oxidized in the presence of metaperiodate,preferably in the presence of sodium periodate (NaIO₄). In anotherembodiment the capsular polysaccharide is oxidized in the presence oforthoperiodate, preferably in the presence of periodic acid.

In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxideradical compound, such as piperidine-N-oxy or pyrrolidine-N-oxycompounds, in the presence of an oxidant to selectively oxidize primaryhydroxyls (as described in WO 2014/097099). In said reaction, the actualoxidant is the N-oxoammonium salt, in a catalytic cycle. In an aspect,said stable nitroxyl or nitroxide radical compound are piperidine-N-oxyor pyrrolidine-N-oxy compounds. In an aspect, said stable nitroxyl ornitroxide radical compound bears a TEMPO(2,2,6,6-tetramethyl-1-piperidinyloxy) or a PROXYL(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In an aspect, saidstable nitroxyl radical compound is TEMPO or a derivative thereof. In anaspect, said oxidant is a molecule bearing a N-halo moiety. In anaspect, said oxidant is selected from the group consisting ofN-Chlorosuccinimide, N-Bromosuccinimide, N-lodosuccinimide,Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.Preferably said oxidant is N-Chlorosuccinimide.

In certain aspects, the oxidizing agent is2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) free radical andN-Chlorosuccinimide (NCS) as the cooxidant (as described in WO2014/097099). Therefore in one aspect, the glycoconjugates from S.pneumoniae are obtainable by a method comprising the steps of: a)reacting a saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)and N-chlorosuccinimide (NCS) in an aqueous solvent to produce anactivated saccharide; and b) reacting the activated saccharide with acarrier protein comprising one or more amine groups (said method isdesignated “TEMPO/NCS-reductive amination” thereafter).

Optionally the oxidation reaction is quenched by addition of a quenchingagent. The quenching agent maybe selected from vicinal diols,1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid (such as glycerol, ethylene glycol, propan-1,2-diol,butan-1,2-diol or butan-2,3-diol, ascorbic acid).

In certain embodiments, the instant invention provides a method forpreparing a serotype 8 Streptococcus pneumoniae polysaccharide-proteinconjugate utilizing a conjugation reaction in an aprotic solvent,wherein the conjugation reaction does not use cyanoborohydride. Infurther embodiments, the conjugation reaction is a Schiff base reductionor reductive amination. In further embodiments, the protein is tetanustoxoid, diphtheria toxoid, or CRM197. In still further embodiments theprotein is CRM197. In further embodiments, the conjugation reaction isreductive amination. In further embodiments, the reductive amination isperformed in dimethylsulfoxide (DMSO).

In some embodiments, the oxidized polysaccharides before conjugationhave a molecular weight of between 30 kDa and 1,000 kDa. Molecularweight can be calculated by size exclusion chromatography (SEC) combinedwith multiangle light scattering detector (MALS) and refractive indexdetector (RI). In some embodiments, the polysaccharide has a molecularweight of between 50 kDa and 300 kDa. In some embodiments, thepolysaccharide has a molecular weight of between 50 kDa and 1,000 kDa.In additional embodiments, the polysaccharide has a molecular weight ofbetween 70 kDa and 900 kDa. In other embodiments, the polysaccharide hasa molecular weight of between 100 kDa and 800 kDa. In other embodiments,the polysaccharide has a molecular weight of between 200 kDa and 600kDa. In further embodiments, the polysaccharide has a molecular weightof 100 kDa to 1,000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDato 700 kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa;100 kDa to 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to800 kDa; 150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150kDa to 400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900kDa; 200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDato 500 kDa; 200 kDa to 400 kDa; 200 kDa to 300; 250 kDa to 1,000 kDa;250 kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDa to600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300kDa to 1,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to 700kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDato 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700kDa; 400 kDa to 600 kDa; or 500 kDa to 600 kDa.

The second step of the conjugation process is the reduction of the imine(Schiff base) bond between activated polysaccharide and a carrierprotein to form a stable conjugate bond (so-called reductive amination),using a reducing agent. Reducing agents which are suitable include thecyanoborohydrides (such as sodium cyanoborohydride or sodiumborohydride). In one embodiment the reducing agent is sodiumcyanoborohydride.

In certain embodiments, the reductive amination reaction is carried outin aprotic solvent (or a mixture of aprotic solvents). In oneembodiment, the reduction reaction is carried out in DMSO or in DMF(dimethylformamide) solvent. The DMSO or DMF solvent may be used toreconstitute the activated polysaccharide and carrier protein, iflyophilized. In one embodiment, the aprotic solvent is DMSO.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, which may be capped using a suitablecapping agent. In one embodiment this capping agent is sodiumborohydride (NaBH₄). Suitable alternatives include sodiumtriacetoxyborohydride or sodium or zinc borohydride in the presence ofBronsted or Lewis acids), amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMe′PrN—BH₃, benzylamine-BH₃ or 5-ethyl-2-methylpyridine borane(PEMB) or borohydride exchange resin. Following the conjugation (thereduction reaction and optionally the capping), the glycoconjugates maybe purified (enriched with respect to the amount ofpolysaccharide-protein conjugate) by a variety of techniques known tothe skilled person. These techniques include dialysis,concentration/diafiltration operations, tangential flow filtration,precipitation/elution, column chromatography (ion exchangechromatography, multimodal ion exchange chromatography, DEAE, orhydrophobic interaction chromatography), and depth filtration. In anembodiment, the glycoconjugates are purified by diafilitration or ionexchange chromatography or size exclusion chromatography.

Glycoconjugates prepared using reductive amination in an aprotic solventare generally used in multivalent pneumococcal conjugate vaccines. Thus,in certain embodiments for multivalent compositions where not all theserotypes are prepared in an aprotic solvent, the reduction reaction forthe remaining seroytpes is carried out in aqueous solvent (e.g.,selected from PBS (phosphate buffered saline), MES(2-(N-morpholino)ethanesulfonic acid), HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-tris, ADA(N-(2-Acetamido)iminodiacetic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPSO(3-Morpholino-2-hydroxypropanesulfonic acid), BES(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), DIPSO (3-Bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS(4-(N-morpholino)butanesulfonic acid), HEPPSO(N-(2-Hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)), POPSO(Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA(triethanolamine), EPPS (4-(2-Hydroxyethyl)piperazine-1-propanesulfonicacid), or Bicine at a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and7.5).

S. pneumoniae capsular polysaccharide-protein conjugates that can beprepared using reductive amination in an aprotic solvent, include, butare not limited to, S. pneumoniae serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F,8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F,24F, 33F and 35B. The polysaccharides may be used in the form ofoligosaccharides. These are conveniently formed by fragmentation ofpurified polysaccharide (e.g. by hydrolysis), which will usually befollowed by purification of the fragments of the desired size.

In certain embodiments, pneumococcal polysaccharide-protein conjugatesof one or more of the S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C,7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B,23F, 24F, 33F and 35B are prepared using reductive amination in anaprotic solvent. In certain embodiments, each of the conjugates in themultivalent immunogenic composition is prepared using reductiveamination in an aprotic solvent. In certain embodiments, polysaccharidesof one or more serotypes in a multivalent composition of the inventionare conjugated to a carrier protein using reductive amination in anaprotic solvent and polysaccharides of one or more serotypes areconjugated using reductive amination in an aqueous solvent. In certainembodiments, polysaccharides of two or more serotypes in a multivalentcomposition of the invention are conjugated to a carrier protein usingreductive amination in an aprotic solvent. In other embodiments,polysaccharides of three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, eleven ormore, twelve or more, thirteen or more, fourteen or more, fifteen ormore, sixteen or more, seventeen or more, eighteen or more, nineteen ormore, twenty or more, twenty-one or more, twenty-two or more,twenty-three or more, or twenty-four or more serotypes in a multivalentcomposition of the invention are conjugated to a carrier protein usingreductive amination in an aprotic solvent. In certain embodiments,polysaccharides from one or more serotypes in a multivalent compositionof the invention are conjugated to a carrier protein using otherchemistries which may be in an aprotic solvent or in an aqueous solvent.

Thus, the invention relates to a multivalent immunogenic compositioncomprising multiple S. pneumoniae polysaccharide protein conjugates,each comprising capsular polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotypes of S. pneumoniaeare as described herein (i.e. in Section II, “Multivalent ImmunogenicCompositions”), wherein the conjugation reaction whereby the S.pneumonia polysaccharide of one or more of the polysaccharide proteinconjugates is conjugated to the carrier protein is in an aproticsolvent. In certain embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the pneumococcalserotypes in a multivalent immunogenic composition are conjugated in anaprotic solvent. The remainder of the serotypes are conjugated using analternative chemistry and/or in an aqueous solvent.

It was determined that the use of DMSO as a solvent during reductiveamination of polysaccharide-protein conjugates results in theunexpectedly superior stability and enhanced immunogenicity for thoseserotypes relative to the same conjugates prepared under aqueousconditions (See US Application Serial No's. 62/463,216 and 62/555,444).

In certain embodiments of the invention the total polysaccharideconcentration in the composition is from about 0.02 to about 0.288mg/mL. In certain embodiments of the invention the total polysaccharideconcentration in the composition is from about 0.03 to about 0.192mg/mL. In certain embodiments of the invention the total polysaccharideconcentration in the composition is from about 0.04 to about 0.192mg/mL. In other embodiments, the total polysaccharide concentration inthe composition is from about 0.065 to about 0.096 mg/mL, about 0.070 toabout 0.080 mg/mL, about 0.065 to about 0.080 mg/mL, about 0.070 toabout 0.085 mg/mL, about 0.110 to about 0.128 mg/mL, about 0.110 toabout 0.175 mg/mL, about 0.10 to about 0.175 mg/mL, about 0.110 to about0.170 mg/mL, about 0.115 to about 0.15 mg/mL, about 0.110 to about 0.15mg/mL, about 0.110 to about 0.125 mg/mL, about 0.150 to about 0.170mg/mL, about 0.150 to about 0.165 mg/mL, about 0.140 to about 0.170mg/mL, about 0.130 to about 0.170 mg/mL, about 0.150 to about 0.175mg/mL, about 0.070 to about 0.170 mg/mL, about 0.065 to about 0.175mg/mL, or about 0.065 to about 0.180 mg/mL.

In embodiments of the invention wherein one or more, or all, of thepolysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the totalpolysaccharide concentration in the composition is stable for 4 weeks ormore at 37° C., 4 weeks or more at 25° C., or 12 weeks or more at 4° C.

In certain embodiments of the invention wherein one or more, or all, ofthe polysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the average molecularweight (Mw) of all of the S. pneumoniae polysaccharide proteinconjugates in the composition (average of all conjugates in thecomposition) is from about 2,000 to about 6,500 kDa, from about 2,500 toabout 6,000 kDa, from about 3,000 to about 5,500 kDa, from about 3,500to about 5,000 kDa, from about 3,500 to about 4,500 kDa, from about3,500 to about 4,700 kDa, from about 3,500 to about 4,600 kDa, fromabout 3,500 to about 4,500 kDa, from about 3,500 to about 4,400 kDa,from about 3,500 to about 4,300 kDa, from about 3,500 to about 4,200kDa, from about 3,600 to about 4,700 kDa, from about 3,600 to about4,600 kDa, from about 3,600 to about 4,500 kDa, from about 3,600 toabout 4,400 kDa, from about 3,600 to about 4,300 kDa, from about 3,600to about 4,200 kDa, from about 3,700 to about 4,700 kDa, from about3,700 to about 4,600 kDa, from about 3,700 to about 4,500 kDa, fromabout 3,700 to about 4,400 kDa, from about 3,700 to about 4,300 kDa,from about 3,700 to about 4,200 kDa, from about 3,800 to about 4,700kDa, from about 3,800 to about 4,600 kDa, from about 3,800 to about4,500 kDa, from about 3,800 to about 4,400 kDa, from about 3,800 toabout 4,300 kDa, from about 3,800 to about 4,200 kDa, from about 3,900to about 4,700 kDa, from about 3,900 to about 4,600 kDa, from about3,900 to about 4,500 kDa, from about 3,900 to about 4,400 kDa, fromabout 3,900 to about 4,300 kDa, or from about 3,900 to about 4,200 kDa.

In certain embodiments of the invention wherein thepolysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the Mw of each of theS. pneumoniae polysaccharide protein conjugates in the composition (fora single serotype) is from about 1,000 to about 10,000 kDa, from about1,500 to about 5,500 kDa, from about 1,500 to about 5,600 kDa, fromabout 1,500 to about 5,700 kDa, from about 1,500 to about 5,800 kDa,from about 1,500 to about 5,900 kDa, from about 1,500 to about 6,000kDa, from about 1,000 to about 5,500 kDa, from about 1,000 to about5,000 kDa, from about 1,000 to about 4,000 kDa, from about 1,000 toabout 4,500 kDa, from about 1,000 to about 4,000 kDa, or from about1,000 to about 3,500 kDa. In other embodiments, the Mw of a conjugatefrom a single serotype within the composition is about 1,000 kDa, about1,100 kDa, about 1,200 kDa, about 1,300 kDa, about 1,400 kDa, about1,500 kDa, about 1,600 kDa, about 1,700 kDa, about 1,800 kDa, about1,900 kDa, about 2,000 kDa, about 2,100 kDa, about 2,200 kDa, about2,300 kDa, about 2,400 kDa, about 2,500 kDa, about 2,600 kDa, about2,700 kDa, about 2,800 kDa, about 2,900 kDa, about 3,000 kDa, about3,100 kDa, about 3,200 kDa, about 3,300 kDa, about 3,400 kDa, about3,500 kDa, about 3,600 kDa, about 3,700 kDa, about 3,800 kDa, about3,900 kDa, about 4,000 kDa, about 4,100 kDa, about 4,200 kDa, about4,300 kDa, about 4,400 kDa, about 4,500 kDa, about 4,600 kDa, about4,700 kDa, about 4,800 kDa, about 4,900 kDa, about 5,000 kDa, about5,100 kDa, about 5,200 kDa, about 5,300 kDa, about 5,400 kDa, or about5,500 kDa.

In certain embodiments of the invention the polysaccharide-proteinconjugates in the multivalent immunogenic compositions are prepared inan aprotic solvent. In certain embodiments, the percentage (ascalculated by the number of polysaccharide serotypes prepared in anaprotic solvent divided by the total number of polysaccharide serotypes,where total number includes those prepared in an aprotic solvent or aprotic solvent) of S. pneumoniae serotype specific conjugates preparedin an aprotic solvent may be greater than 50%, or greater than 60%, orgreater than 70%, or greater than 80%, or greater than 90% or are 100%.

In certain embodiments of the invention, the serotype 3polysaccharide-protein conjugate in the composition is prepared in anaprotic solvent and the Mw of said conjugate is from about 1,000 toabout 5,000 kDa, or from about 1,000 to about 4,000 kDa, or from about1,000 to about 3,000 kDa, or from about 1,000 to about 2,500 kDa, orfrom about 1,000 to about 2,000 kDa.

In certain embodiments of the invention wherein one or more, or all, ofthe polysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the number averagemolecular weight (Mn) of the S. pneumoniae polysaccharide proteinconjugates in the composition (average of all conjugates in thecomposition) is from about 900 to about 3,000 kDa, from about 1,000 toabout 3,000 kDa, from about 1,000 to about 2,500 kDa, from about 1,500to about 2,500 kDa, from about 1,800 to about 2,500 kDa, from about1,900 to about 2,500 kDa, or from about 2,000 to about 2,500 kDa.

In certain embodiments of the invention wherein one or more, or all, ofthe polysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the Mn of each of theS. pneumoniae polysaccharide protein conjugates in the composition (fora single serotype) is from about 700 to about 7,000 kDa, from about1,000 to about 6,000 kDa, from about 1,000 to about 5,000 kDa, fromabout 1,000 to about 4,000 kDa, from about 1,000 to about 3,000 kDa,from about 900 to about 5,500 kDa, from about 900 to about 5,000 kDa,from about 900 to about 4,500 kDa, from about 900 to about 4,000 kDa,from about 900 to about 3,500 kDa, or from about 900 to about 3,000 kDa.

In embodiments of the invention, the Mw and/or Mn of the S. pneumoniaepolysaccharide protein conjugates in the composition is stable for 4weeks or more at 37° C., 4 weeks or more at 25° C., and/or 12 weeks ormore at 4° C.

In embodiments of the invention, the polysaccharide concentration, Mw,and/or Mn are determined using HPSEC UV/MALS/RI.

In some embodiment of the invention, wherein one or more, or all, of thepolysaccharide-protein conjugates in the multivalent immunogeniccompositions are prepared in an aprotic solvent, the emission maximum ofthe composition measured using intrinsic protein fluorescencespectroscopy with an excitation wavelength at 280 nanometers (nm) isfrom about 335 nm to about 342 nm. In some embodiments, the emissionmaximum remains from about 335 nm to about 342 nm and the fluorescenceintensity is stable for at least 1 week at 37° C. In some embodiments,the emission maximum remains from about 335 nm to about 342 nm and thefluorescence intensity is stable for 1 week at 37° C.

In some embodiments, all of the pneumococcal polysaccharide conjugatesin the multivalent composition are prepared using reductive amination inDMSO. In certain sub-embodiments, the multivalent composition comprisingpolysaccharide conjugates which were all prepared using DMSO does notcomprise an adjuvant.

Without being bound by any particular theory, one possible mechanism forthe enhanced immunogenicity observed with glycoconjugates prepared inDMSO include an increased number of linkages between the carbohydrate(capsular polysaccharide) and lysine residues on the surface of thecarrier protein which would result in additional attachment pointsbetween the protein and polysaccharide to impart stability and counterchemical depolymerization or breakdown of the peptide carbohydrate bond.See, e.g., Hsieh, Characterization of Saccharide-CRM197 ConjugateVaccines in Brown F, Corbel M, Griffiths E (eds): Physico-ChemicalProcedures for the Characterization of Vaccines. Dev. Biol. Basel,Karger, 2000, vol 103, pp. 93-104. An additional benefit of theincreased polysaccharide-protein linkages that are created duringconjugation in the DMSO solvent could be additional opportunities forsuccessful presentation of peptide-carbohydrate to T-cells. A possiblemechanism of enhanced immunogenicity observed by conjugation in the DMSOsolvent could be due to the denaturation of CRM197 in organic solvent,which exposes additional lysines for polysaccharide linkages givingincreased chances for glycopeptide presentation at the surface of an APCfor T-cell dependent response to different peptide epitopes. See Avci etal., 2011, Nature Medicine 17: 1602-1610.

Yet another benefit of conjugation in an organic solvent generatingdenatured CRM197 in the conjugates could be reduced immunologicalinterference of antibodies against native CRM197 epitopes. A furtherbenefit of the increased polysaccharide-protein linkages that arecreated during conjugation in the DMSO solvent could be the formation oflarger sized polysaccharide protein conjugates resulting in enhancedimmunogenicity. The compositions of the invention are believed toprovide significant advantages in eliciting a human response.

In certain embodiments, the conjugation reaction is performed byreductive amination wherein nickel is used for greater conjugationreaction efficiency and to aid in free cyanide removal. Transitionmetals are known to form stable complexes with cyanide and are known toimprove reductive methylation of protein amino groups and formaldehydewith sodium cyanoborohydride (S Gidley et al., Biochem J 1982, 203:331-334; Jentoft et al. Anal Biochem. 1980, 106: 186-190). By complexingresidual, inhibitory cyanide, the addition of nickel increases theconsumption of protein during the conjugation and leads to formation oflarger, potentially more immunogenic conjugates.

Differences in starting cyanide levels in sodium cyanoborohydridereagent lots also lead to inconsistent conjugation performance,resulting in variable product attributes, such as conjugate size andconjugate Ps-to-CRM197 ratio. The addition of nickel reduced conjugationinconsistency by complexing cyanide, eliminating differences in sodiumcyanoborohydride lots.

Suitable alternative chemistries include the activation of thesaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester. The activated saccharide may thus becoupled directly or via a spacer (linker) group to an amino group on thecarrier protein. For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example using GMBS) or ahaloacetylated carrier protein (for example using iodoacetimide [e.g.ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB, or SIA,or SBAP). Preferably, the cyanate ester (optionally made by CDAPchemistry) is coupled with hexane diamine or adipic acid dihydrazide(ADH) and the amino-derivatised saccharide is conjugated to the carrierprotein using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxylgroup on the protein carrier. Such conjugates are described inInternational Patent Application Publication Nos. WO 93/15760, WO95/08348 and WO 96/29094; and Chu et al., 1983, Infect. Immunity40:245-256.

Other suitable conjugation techniques use carbodiimides, hydrazides,active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,S-NHS, EDC, TSTU. Many are described in International Patent ApplicationPublication No. WO 98/42721. Conjugation may involve a carbonyl linkerwhich may be formed by reaction of a free hydroxyl group of thesaccharide with CDI (See Bethell et al., 1979, J. Biol. Chem.254:2572-4; Hearn et al., 1981, J. Chromatogr. 218:509-18) followed byreaction with a protein to form a carbamate linkage. This may involvereduction of the anomeric terminus to a primary hydroxyl group, optionalprotection/deprotection of the primary hydroxyl group, reaction of theprimary hydroxyl group with CDI to form a CDI carbamate intermediate andcoupling the CDI carbamate intermediate with an amino group on aprotein.

After conjugation of the capsular polysaccharide to the carrier protein,the polysaccharide-protein conjugates are purified (enriched withrespect to the amount of polysaccharide-protein conjugate) by one ormore of a variety of techniques. Examples of these techniques are wellknown to the skilled artisan and include concentration/diafiltrationoperations, ultrafiltration, precipitation/elution, columnchromatography, and depth filtration. See, e.g., U.S. Pat. No.6,146,902.

After the individual glycoconjugates are purified, they are compoundedto formulate the immunogenic composition of the present invention. Thesepneumococcal conjugates may be prepared by separate processes and bulkformulated into a single dosage formulation.

An alternative method for characterizing the glycoconjugates of theinvention is by the number of lysine residues in the carrier protein(e.g., CRM197) that become conjugated to the saccharide which can becharacterized as a range of conjugated lysines (degree of conjugation).The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, can be obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered, compared to the carrier protein starting material used togenerate the conjugate materials. In a preferred embodiment, the extentof conjugation, as measured by lysine consumption of the glycoconjugateof the invention is between 2 and 15, between 2 and 13, between 2 and10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4,between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8,between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15,between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15or between 10 and 12. In an embodiment, the degree of conjugation of theglycoconjugate of the invention is about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14 or about 15. In another embodiment, the degree ofconjugation of the glycoconjugate of the invention is between 4 and 7.In some such embodiments, the carrier protein is CRM197.

The glycoconjugates of the compositions of the invention may also becharacterized by the ratio (weight/weight) of polysaccharide to carrierprotein (Ps:Pr). In some embodiments, the ratio of polysaccharide tocarrier protein of the glycoconjugates (w/w) in the composition isbetween 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7, about 0.8,about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1,about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about2.8, about 2.9, or about 3.0). In other embodiments, the polysaccharideto carrier protein ratio (w/w) is between 0.5 and 2.5, between 0.5 and1.5, between 0.8 and 2.5, between 0.5 and 1.0, between 1.0 and 1.5,between 1.0 and 2.0, between 0.8 and 2.4, between 0.8 and 2.3, between0.8 and 2.2, between 0.8 and 2.1, between 0.8 and 2.0, between 0.8 and1.9, between 0.8 and 1.8, between 0.8 and 1.7, between 0.8 and 1.6,between 0.8 and 1.5, between 0.8 and 1.4, between 0.8 and 1.3, between0.9 and 2.4, between 0.9 and 2.3, between 0.9 and 2.2, between 0.9 and2.1, between 0.9 and 2.0, between 0.9 and 1.9, between 0.9 and 1.8,between 0.9 and 1.7, between 0.9 and 1.6, between 0.9 and 1.5, between0.9 and 1.4, between 0.9 and 1.3, between 0.9 and 1.2, between 1.0 and2.4, between 1.0 and 2.3, between 1.0 and 2.2, between 1.0 and 2.1,between 1.0 and 2.0, between 1.0 and 1.9, between 1.0 and 1.8, between1.0 and 1.7, between 1.0 and 1.6, between 1.0 and 1.5, between 1.0 and1.4, between 1.0 and 1.3 or between 1.0 and 1.2. In further embodiments,the saccharide to carrier protein ratio (w/w) is between 0.8 and 1.2. Insome such embodiments, the carrier protein is CRM197. Theglycoconjugates and immunogenic compositions of the invention maycontain free saccharide that is not covalently conjugated to the carrierprotein, but is nevertheless present in the glycoconjugate composition.The free saccharide may be non-covalently associated with (e.g.,non-covalently bound to, adsorbed to, or entrapped in or with) theglycoconjugate.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 15A conjugate is from about 1.0 to about 2.0, fromabout 1.25 to about 1.75, or from about 1.3 to about 1.7. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype15A is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 15C conjugate is from about 1.0 to about 2.0, fromabout 1.25 to about 1.75, or from about 1.3 to about 1.7. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype15C is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 33F conjugate is from about 1.0 to about 2.0, fromabout 1.25 to about 1.75, or from about 1.3 to about 1.7. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype33F is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 35B conjugate is from about 1.25 to about 2.25, fromabout 1.25 to about 2.0, or from about 1.3 to about 1.8. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype35B is about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0.

In specific embodiments, the saccharide to carrier protein ratio (w/w)for the serotype 24F conjugate is from about 0.5 to about 1.5, fromabout 0.75 to about 1.25, or from about 0.8 to about 1.0. In otherembodiments, the saccharide to carrier protein ratio (w/w) for serotype24F is about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

In a preferred embodiment, the glycoconjugate composition comprises lessthan about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of freepolysaccharide compared to the total amount of polysaccharide. In apreferred embodiment the glycoconjugate composition comprises less thanabout 25% of free polysaccharide compared to the total amount ofpolysaccharide. In a preferred embodiment the glycoconjugate compositioncomprises less than about 20% of free polysaccharide compared to thetotal amount of polysaccharide. In a preferred embodiment theglycoconjugate composition comprises less than about 15% of freepolysaccharide compared to the total amount of polysaccharide.

IV. Methods of Use

Embodiments of the invention also include one or more of the multivalentimmunogenic compositions described herein (i) for use in, (ii) for useas a medicament or composition for, or (iii) for use in the preparationof a medicament for: (a) therapy (e.g., of the human body); (b)medicine; (c) inhibition of infection with Streptococcus pneumoniae; (d)induction of an immune response or a protective immune response againstS. pneumoniae; (e) prophylaxis of infection by S. pneumoniae; (f)prevention of recurrence of S. pneumoniae infection; (g) reduction ofthe progression, onset or severity of pathological symptoms associatedwith S. pneumoniae infection including the prevention of associatedcomplications such as brain damage, hearing loss, and seizures, (h)reduction of the likelihood of a S. pneumoniae infection or, (i)treatment, prophylaxis of, or delay in the onset, severity, orprogression of pneumococcal disease(s), including, but not limited to:pneumococcal pneumonia, pneumococcal bacteremia, pneumococcalmeningitis, otits media and sinusitis. In these uses, the multivalentpneumococcal polysaccharide-conjugate compositions of the invention canoptionally be employed in combination with one or more adjuvants, orwithout an adjuvant.

Accordingly, the invention provides methods for the prophylactictreatment of (i.e. protection against) S. pneumoniae infection orpneumococcal disease comprising administering one or more of themultivalent immunogenic pneumococcal polysaccharide-protein conjugatecompositions of the invention to a patient in need of treatment.

The compositions and formulations of the present invention can be usedto protect or treat a human susceptible to infection, e.g., apneumococcal infection, by means of administering such composition orformulation via a systemic or mucosal route.

In one embodiment, the invention provides a method of inducing an immuneresponse to S. pneumoniae, comprising administering to a patient animmunologically effective amount of a multivalent immunogeniccomposition of the invention. In another embodiment, the inventionprovides a method of vaccinating a human against a pneumococcalinfection, comprising the step of administering to the human animmunogically effective amount of a multivalent immunogenic compositionof the invention.

Thus, in one aspect, the invention provides a method for (1) inducing animmune response in a human patient, (2) inducing a protective immuneresponse in a human patient, (3) vaccinating a human patient against aninfection with S. pneumoniae, or (4) reducing the likelihood of a S.pneumoniae infection in a human patient, the method comprisingadministering a multivalent immunogenic composition of the invention tothe patient (i.e. any multivalent immunogenic composition describedherein, such as the multivalent immunogenic compositions described inSection II, entitled “Multivalent Immunogenic Compositions,” supra).

In one embodiment, the invention provides a method for the prevention ofpneumococcal pneumonia and/or invasive pneumococcal disease in an infant(less than 1 year of age), toddler (approximately 12 to 24 months), oryoung child (approximately 2 to 5 years).

In another embodiment, the invention provides a method for theprevention of pneumococcal pneumonia and/or invasive pneumococcaldisease in a 6 week through 17 year old patient.

In another embodiment, the invention provides a method for theprevention of pneumococcal pneumonia and/or invasive pneumococcaldisease in a 6 month through 17 year old patient.

In another embodiment, the invention provides a method for theprevention of pneumococcal pneumonia and/or invasive pneumococcaldisease in adults 18 years of age and older.

In another embodiment, the invention provides a method for theprevention of pneumococcal pneumonia and/or invasive pneumococcaldisease in adults 50 years of age and older.

In another embodiment, the invention provides a method for theprevention of pneumococcal pneumonia and/or invasive pneumococcaldisease in adults 65 years of age and older.

In another embodiment, the invention provides a method for theprevention of pneumococcal pneumonia and/or invasive pneumococcaldisease caused by one or more of the following Streptococcus pneumoniaestrains: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In one embodiment of the methods above, the composition comprisesmultiple S. pneumoniae polysaccharide protein conjugates wherein each ofthe conjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotypes of S. pneumoniaecomprise serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 12F, 14,15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B orserotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A,15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In anotherembodiment of the methods above, the composition comprises multiple S.pneumoniae polysaccharide protein conjugates wherein each of theconjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotypes of S. pneumoniaeconsist of serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 12F, 14,15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B orserotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A,15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In oneembodiment of the methods above, the composition comprises multiple S.pneumoniae polysaccharide protein conjugates wherein each of theconjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotypes of S. pneumoniaecomprise serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A,15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or serotypes: 1, 3,4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F and 35B. In another embodiment of the methodsabove, the composition comprises multiple S. pneumoniae polysaccharideprotein conjugates wherein each of the conjugates comprises apolysaccharide from an S. pneumoniae serotype conjugated to a carrierprotein, wherein the serotypes of S. pneumoniae consist of serotypes: 1,3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,23B, 23F, 24F, 33F and 35B or serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B. In one embodiment of the methods above, the composition comprisesmultiple S. pneumoniae polysaccharide protein conjugates wherein each ofthe conjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotypes of S. pneumoniaecomprise serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C,18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. In another embodiment ofthe methods above, the composition comprises multiple S. pneumoniaepolysaccharide protein conjugates wherein each of the conjugatescomprises a polysaccharide from an S. pneumoniae serotype conjugated toa carrier protein, wherein the serotypes of S. pneumoniae consist ofserotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,19F, 22F, 23B, 23F, 24F, 33F and 35B or serotypes:

It has been shown that a pneumococcal conjugate vaccine comprisingserotype 6A polysaccharide may provide some cross-protection againstserotype 6C (Cooper et al., Vaccine 29 (2011) 7207-7211). Therefore, insome embodiments of the methods above, the invention also provides useof multivalent immunogenic compositions that do not comprise serotype 6Cpolysaccharide conjugate, but instead comprise serotype 6Apolysaccharide conjugate or serotypes 6A and 6B polysaccharideconjugates. In other embodiments, the immunogenic composition comprisespneumococcal polysaccharide conjugates of serotypes 6A, 6B, and 6C.

In particular embodiments of the methods above, the multivalentimmunogenic compositions comprise pneumococcal conjugates that includepolysaccharides of a group of S. pneumoniae serotypes selected from thegroup consisting of:

-   -   a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A,        DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;    -   z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; and    -   aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In further embodiments of the methods above, the composition comprisesmultiple S. pneumoniae polysaccharide protein conjugates wherein each ofthe conjugates comprises a polysaccharide from an S. pneumoniae serotypeconjugated to a carrier protein, wherein the serotypes of S. pneumoniaecomprise serotypes: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A,15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5,6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,24F, 33F and 35B; or iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F,14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or iv) 1,3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F and 35B.

It has also been shown that a pneumococcal conjugate vaccine comprisingserotype 10A polysaccharide may provide some cross-protection againstserotype 39 (see WO 2017/085586). Therefore, in some embodiments of themethods above, the invention also provides use of multivalentimmunogenic compositions that do not comprise serotype 10Apolysaccharide conjugate, but instead comprise serotype 39polysaccharide conjugate. In other embodiments, the immunogeniccomposition comprises pneumococcal polysaccharide conjugates ofserotypes 10A and 39. In particular embodiments of the methods above,the serotypes of S. pneumoniae comprise a group of serotypes selectedfrom the group consisting of:

-   -   bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, DeOAc15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   cc) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   dd) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, DeOAc15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, DeOAc15B,        18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ll) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   nn) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   qq) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   rr) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   tt) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   uu) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   ww) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F,        22F, 23B, 23F, 24F, 33F, 35B and 39;    -   zz) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A,        19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;    -   aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C,        19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; and bbb) 1, 3, 4,        5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,        23B, 23F, 24F, 33F, 35B and 39.

It has also been shown that immunogenic conjugates comprising S.pneumoniae serotype 15B capsular polysaccharide covalently linked to acarrier protein may provide some cross-protection against serotype 15Cand/or serotype 15A (see WO 2015/110942). Therefore, in some embodimentsof the methods above, the invention also provides use of multivalentimmunogenic compositions that do not comprise serotype 15C (orde-O-acetylated 15B) polysaccharide conjugate, but instead compriseserotype 15B polysaccharide conjugate (i.e. the serotype 15Bpolysaccharide is not substantially de-O-acetylated). In otherembodiments, the immunogenic composition comprises pneumococcalpolysaccharide conjugates of serotypes 15B and 15C (or de-O-acetylated15B).

The compositions of the invention are useful in methods for providingcomplementary protection against S. pneumoniae in patients who hadpreviously received a multivalent pneumococcal vaccine. In this use, thecompositions of the invention can provide protection against particularS. pneumoniae serotypes that a patient had not been previouslyvaccinated against, can provide additional protection against S.pneumoniae serotypes that a patient had been previously vaccinatedagainst, or can provide protection against both S. pneumoniae serotypesthat a patient had not been previously vaccinated against and S.pneumoniae serotypes that a patient had been previously vaccinatedagainst.

Thus, the invention provides a method of inducing an immune response,vaccinating, or inducing a protective immune response against S.pneumoniae in a patient, comprising administering a multivalentimmunogenic composition to the patient, the composition comprisingmultiple S. pneumoniae polysaccharide protein conjugates, wherein thepolysaccharide protein conjugates comprise capsular polysaccharide froma S. pneumoniae serotype conjugated to a carrier protein, wherein thepatient had previously been vaccinated against S. pneumoniae. Inembodiments of this aspect of the invention, the multivalent immunogeniccomposition can be any multivalent immunogenic composition describedherein. In particular embodiments of the methods of the invention, themultivalent immunogenic composition is administered to a patient who waspreviously treated with a multivalent pneumococcal vaccine. Themultivalent immunogenic vaccine may be any vaccine that is indicated forthe prevention of pneumococcal disease caused by more than one serotypeof S. pneumoniae.

In specific embodiments of the method above, the patient was previouslytreated with a multivalent pneumococcal vaccine that is indicated forthe prevention of pneumococcal disease caused by one or more S.pneumoniae serotypes within a group of serotypes selected from the groupconsisting of:

-   -   i. 4, 6B, 9V, 14, 18C, 19F and 23F;    -   ii. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, and 19A;    -   iii. 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F;    -   iv. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, and        33F;    -   v. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,        8, 9N, 10A, 11A, 12F, 15B, 17F, and 20; and    -   vi. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F,        33F, 8, 10A, 11A, 12F and 15B.

In specific embodiments of the method above, the multivalentpneumococcal vaccine comprises multiple polysaccharide proteinconjugates, wherein the polysaccharide protein conjugates comprisepolysaccharide from a S. pneumoniae serotype conjugated to a carrierprotein. In other embodiments, the multivalent pneumococcal vaccinecomprises multiple S. pneumoniae capsular polysaccharides that are notconjugated to a carrier protein.

In additional embodiments of the method above, the patient waspreviously treated with PREVNAR® 13 (Pneumococcal 13-valent ConjugateVaccine [Diphtheria CRM197 Protein], Pfizer, Inc., Philadelphia, Pa.,USA).

In further embodiments of the method above, the patient was previouslytreated with PNEUMOVAX® 23 (Pneumococcol Vaccine Polyvalent, Merck &Co., Inc., Kenilworth, N.J., USA).

In still further embodiments of the method above, the patient waspreviously treated with SYNFLORIX™ (Pneumococcal polysaccharideconjugate vaccine (adsorbed), GlaxoSmithKline Biologicals s.a.,Rixensart, Belgium).

In embodiments of the method above, the multivalent immunogeniccomposition of the invention is administered to a patient at any timeafter the patient has received a multivalent pneumococcal vaccine,according to the treatment regimen provided by the medical professional,e.g. a physician. In particular embodiments, the multivalent immunogeniccomposition of the invention is administered to a patient from about 1month to about 5 years after the patient has received the multivalentpneumococcal vaccine, alternatively, from 1 month to 1 year, from 1month to 2 years, from 1 month to 3 years, from 1 month to 4 years, from1 month to 6 months, from 2 months to 6 months, from 2 months to 1 year,from 1 year to 5 years, from 6 months to 5 years, from 6 months to 4years, from 6 months to 3 years, from 6 months to 2 years, from 6 monthsto 1 year, from 1 year to 4 years, from 1 year to 3 years, or from 1year to 2 years, after the patient has received the multivalentpneumococcal vaccine. In further embodiments, the multivalentimmunogenic composition is administered to the patient about 1 month,about 2 months, about 3 months, about 4 months, about 5 months, about 6months, about 7 months, about 8 months, about 9 months, about 10 months,about 11 months, about 1 year, about 1.25 years, about 1.5 years, about1.75 years, about 2 years, about 2.25 years, about 2.5 years, about 2.75years, about 3 years, about 3.25 years, about 3.5 years, about 3.75years, about 4 years, about 4.25 years, about 4.5 years, about 4.75years, or about 5 years after the patient has received the multivalentpneumococcal vaccine.

In further embodiments, the invention provides a method for (1) inducingan immune response in a human patient, (2) inducing a protective immuneresponse in a human patient, (3) vaccinating a human patient against aninfection with S. pneumoniae, or (4) reducing the likelihood of a S.pneumoniae infection in a human patient, the method comprisingadministering a multivalent immunogenic composition of the invention andadministering a multivalent pneumococcal vaccine to the patient, in anyorder. For example, the patient is administered a multivalentpneumococcal vaccine first, and the patient is administered amultivalent immunogenic composition of the invention second.Alternatively, the patient is administered a multivalent immunogeniccomposition of the invention first and is administered a multivalentpneumococcal vaccine second. The multivalent pneumococcal vaccine may beany vaccine indicated for the prevention of pneumococcal disease causedby more than one serotype of S. pneumoniae.

In specific embodiments of the method above, the patient is treated witha multivalent immunogenic composition of the invention and a multivalentpneumococcal vaccine that is indicated for the prevention ofpneumococcal disease caused by one or more S. pneumoniae serotypes of agroup of serotypes selected from the group consisting of: i. 4, 6B, 9V,14, 18C, 19F and 23F;

-   -   ii. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, and 19A;    -   iii. 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F;    -   iv. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, and        33F;    -   v. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,        8, 9N, 10A, 11A, 12F, 15B, 17F, and 20; and    -   vi. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F,        33F, 8, 10A, 11A, 12F and 15B.

In specific embodiments of the method above, the multivalentpneumococcal vaccine comprises capsular polysaccharides of S. pneumoniaeserotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2,8, 9N, 10A, 11A, 12F, 15B, 17F, and 20A.

In specific embodiments of the method above, the multivalentpneumococcal vaccine comprises multiple polysaccharide proteinconjugates, wherein the polysaccharide protein conjugates comprisepolysaccharide from a S. pneumoniae serotype conjugated to a carrierprotein. In other embodiments, the multivalent pneumococcal vaccinecomprises multiple S. pneumoniae capsular polysaccharides that are notconjugated to a carrier protein.

In additional embodiments of the method above, the patient is treatedwith a multivalent immunogenic composition of the invention and istreated with PREVNAR® 13 (Pneumococcal 13-valent Conjugate Vaccine[Diphtheria CRM197 Protein], Pfizer, Inc., Philadelphia, Pa., USA), inany order. In one embodiment, the patient is administered PREVNAR® 13first, and the patient is administered a multivalent immunogeniccomposition of the invention second. In alternative embodiments, thepatient is administered a multivalent immunogenic composition of theinvention first and is administered PREVNAR® 13 second.

In further embodiments of the method above, the patient is treated witha multivalent immunogenic composition of the invention and is treatedwith PNEUMOVAX® 23 (pneumococcal vaccine polyvalent, Merck & Co., Inc.,Kenilworth, N.J., USA), in any order. In one embodiment, the patient isadministered PNEUMOVAX® 23 first, and the patient is administered amultivalent immunogenic composition of the invention second. Inalternative embodiments, the patient is administered a multivalentimmunogenic composition of the invention first and is administeredPNEUMOVAX® 23 second.

In still further embodiments of the method above, the patient is treatedwith a multivalent immunogenic composition of the invention and istreated with SYNFLORIX™ (Pneumococcal polysaccharide conjugate vaccine(adsorbed), GlaxoSmithKline Biologicals s.a., Rixensart, Belgium), inany order. In one embodiment, the patient is administered SYNFLORIX™first, and the patient is administered a multivalent immunogeniccomposition of the invention second. In an alternative embodiment, thepatient is administered a multivalent immunogenic composition of theinvention first and is administered SYNFLORIX™ second.

In some embodiments of the method above, the multivalent immunogeniccomposition and the multivalent pneumococcal vaccine are administeredconcurrently. As used herein, “concurrent administration” is not limitedto dosing of two compositions at the same time, but includesadministration one right after the other in any order. In someembodiments, the multivalent immunogenic composition and the multivalentpneumococcal vaccine are administered via intramuscular or subcutaneousadministration into separate anatomical sites, e.g. two different arms.

In some embodiments of the method above, the amount of time betweenadministration of the multivalent immunogenic composition of theinvention and the multivalent pneumococcal vaccine is from about 4 weeksto about 1 year. In alternative embodiments, the amount of time is fromabout 1 month to about 5 years.

In one embodiment, the patient is administered the multivalentpneumococcal vaccine first and the multivalent immunogenic compositionof the invention second. In alternative embodiments, the patient isadministered a multivalent immunogenic composition of the inventionfirst and is administered the multivalent pneumococcal vaccine second.

Also provided is a method of inducing an immune response, vaccinating orinducing a protective immune response against S. pneumoniae in apatient, comprising:

(1) administering a multivalent immunogenic composition of the inventionto the patient,

(2) waiting for a pre-determined amount of time to pass, and

(3) administering a multivalent pneumococcal vaccine to the patient.

In this method, the multivalent immunogenic composition can comprise anycombination of S. pneumoniae polysaccharide protein conjugates set forthherein and the multivalent pneumococcal vaccine can be any vaccineindicated for the prevention of disease caused by more than one serotypeof S. pneumoniae.

Also provided by the invention is a method of inducing an immuneresponse, vaccinating or inducing a protective immune response againstS. pneumoniae in a patient, comprising:

(1) administering a multivalent pneumococcal vaccine to the patient,

(2) waiting for a pre-determined amount of time to pass, and

(3) administering a multivalent immunogenic composition of the inventionto the patient.

In this method, the multivalent immunogenic composition can comprise anycombination of S. pneumoniae polysaccharide protein conjugates set forthherein and the multivalent pneumococcal vaccine can be any vaccineindicated for the prevention of disease caused by more than one serotypeof S. pneumoniae.

In some embodiments of the methods above, the multivalent pneumococcalvaccine comprises multiple S. pneumoniae polysaccharide proteinconjugates, wherein the polysaccharide protein conjugates comprisecapsular polysaccharide from a S. pneumoniae serotype conjugated to acarrier protein. In alternative embodiments, the multivalentpneumococcal vaccine comprises S. pneumoniae capsular polysaccharidesthat are not conjugated to a carrier protein.

In any embodiments of the methods of the invention (i.e. any of themethods described herein), the method may further comprise administeringone or more additional doses of a multivalent immunogenic composition ofthe invention to the patient. In such methods, the patient may havealready received a multivalent pneumococcal vaccine prior to receiving afirst dose of a multivalent immunogenic composition of the invention,supra, or may not have been vaccinated against S. pneumoniae prior toreceiving a multivalent immunogenic composition of the invention. Thus,in one embodiment, a patient who had received a multivalent pneumococcalvaccine indicated for the prevention of pneumococcal disease caused byS. pneumoniae is administered two or more doses of a multivalentimmunogenic composition of the invention. In alternative embodiments, apatient who had not been previously treated with any vaccine indicatedfor the prevention of pneumococcal disease, is administered two or moredoses of a multivalent immunogenic composition of the invention.

In embodiments of the method above, the two or more doses are of thesame multivalent immunogenic composition of the invention. Inalternative embodiments, the two or more doses are of differentmultivalent immunogenic compositions of the invention.

In specific embodiments of any of these methods, the patient isadministered two, three, or four doses of a multivalent immunogeniccomposition of the invention. In particular embodiments, the patient isimmunocompromised (e.g., on an immunosuppressive regimen following astem cell transplant).

In some embodiments, the amount of time between administration of eachdose of multivalent immunogenic composition of the invention is fromabout 4 weeks to about 1 year. In alternative embodiment, the amount oftime between administration of each dose of multivalent immunogeniccomposition of the invention is from about 1 month to about 5 years.

In embodiments of any of the methods of the invention, the patient to betreated with the composition(s) of the invention is a human. In certainembodiments, the human patient is an infant (approximately 6 weeks to 12months). In certain embodiments, the human patient is a toddler(approximately 12 to 24 months), or young child (approximately 2 to 5years). The compositions of this invention are also suitable for usewith older children, adolescents and adults (e.g., aged 18 to 45 years,aged 18 to 50 years, aged 18 to 55 years, aged 18 to 60 years or 18 to65 years). In other embodiments of any of the methods of the invention,the patient is from about 2 to about 18 years of age. In furtherembodiments of any of the methods of the invention, the patient is 18years of age or older.

In further embodiments of the methods of the invention, the patient isan infant and the infant is administered 1, 2, or 3 doses of amultivalent immunogenic composition of the instant invention. The amountof time between administration of each dose can vary, but an example ofa dosing schedule includes administration of a dose at 2 months of age,then another administration of a dose at 4 months of age, and finally afinal administration of a dose at 6 months of age. Another example of anadministration schedule in infants is administration of a dose at 2months of age and then another administration of a dose at 3 months ofage. Another example of an administration schedule in infants isadministration of a dose at 2 months of age and then anotheradministration of a dose at 3 months of age, and finally a finaladministration of a dose at 6 months of age. In further embodiments, aninfant patient can receive an additional “booster” dose of a multivalentimmunogenic composition of the instant invention when the infant becomesa toddler. For example, an infant is dosed at 2 months of age, thenanother administration of a dose at 4 months of age, and finally a finaladministration of a dose at 6 months of age, then, when the infantreaches the age of a toddler, an additional “booster” dose of amultivalent immunogenic composition of the instant invention isadministered between 11 to 15 months of age.

In an embodiment, an infant is administered 2 doses of a multivalentimmunogenic composition of the instant invention.

In an embodiment, an infant is administered 3 doses of a multivalentimmunogenic composition of the instant invention.

In an embodiment, a patient is administered 3 doses of a multivalentimmunogenic composition of the instant invention, wherein the first andsecond doses are administered between 2 and 10 months of age and thethird dose is administered between 11 to 15 months of age.

In an embodiment, a patient is administered 4 doses of a multivalentimmunogenic composition of the instant invention, wherein the first doseis administered at 2 months of age, the second dose is administered at 4months of age, the third dose is administered at 6 months of age and thefourth dose is administered between 11 to 15 months of age.

In further embodiments of the methods of the invention, the humanpatient is elderly. In some embodiments of any of the methods of theinvention, the patient is 50 years of age or older. In some embodimentsof any of the methods of the invention, the patient is 55 years of ageor older. In some embodiments of any of the methods of the invention,the patient is 60 years of age or older. In still further embodiments ofany of the methods of the invention, the patient is 65 years of age orolder. In additional embodiments of any of the methods of the invention,the patient is 70 years of age or older.

In some embodiments of any of the methods of the invention, the patientto be treated with an immunogenic composition of the invention isimmunocompromised.

In some embodiments of any of the methods of the invention, themultivalent immunogenic composition of the invention is administeredconcomitantly with a vaccine against influenza. In certain embodiments,the influenza vaccine is a “senior flu vaccine,” a high dose flu vaccineindicated for the elderly, e.g. persons aged 65 and older.

The invention provides a method for inducing a protective immuneresponse in a patient against a pneumococcal infection comprising thestep of administering to the patient an immunologically effective amountof any of the multivalent immunogenic pneumococcalpolysaccharide-protein conjugate compositions described herein. Optimalamounts of components for a particular vaccine (e.g. a multivalentimmunogenic composition of the invention) can be ascertained by standardstudies involving observation of appropriate immune responses insubjects. For example, in another embodiment, the dosage for humanvaccination is determined by extrapolation from animal studies to humandata. In another embodiment, the dosage is determined empirically.

The methods of the invention can be used for the prevention and/orreduction of primary clinical syndromes caused by microbes, e.g., S.pneumoniae, including both invasive infections (meningitis, pneumonia,and bacteremia), and noninvasive infections (acute otitis media, andsinusitis).

Administration of the compositions of the invention can include one ormore of: injection via the intramuscular, intraperitoneal, intradermalor subcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory or genitourinary tracts. In one embodiment,intranasal administration is used for the treatment of pneumonia orotitis media (as nasopharyngeal carriage of pneumococci can be moreeffectively prevented, thus attenuating infection at its earlieststage). In specific embodiments, the compositions of the invention areadministered to the patient via intramuscular or subcutaneousadministration.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing methodologies and materials thatmight be used in connection with the present invention.

Having described different embodiments of the invention herein withreference to the accompanying drawings, it is to be understood that theinvention is not limited to those precise embodiments, and that variouschanges and modifications may be effected therein by one skilled in theart without departing from the scope or spirit of the invention asdefined in the appended claims.

The following examples illustrate, but do not limit the invention.

Example 1

Preparation of S. pneumoniae Capsular Polysaccharides

Methods of culturing pneumococci are well known in the art. See, e.g.,Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods ofpreparing pneumococcal capsular polysaccharides are also well known inthe art. See, e.g., European Patent No. EP 0 497 524 B1. The processdescribed below generally follows the method described in EuropeanPatent No. EP 0 497 524 B1 and is generally applicable to allpneumococcal serotypes.

Isolates of pneumococcal strains for serotypes 1, 3, 4, 5, 6A, 6B, 7F,8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and35B were obtained from Merck Culture Collection. Strains for serotypes23B were obtained from Centers of Disease Control and Prevention andUniversity of Alabama Birmingham. Strains for serotype 24F were obtainedfrom Merck Culture Collection and University of Alabama Birmingham.Where needed, subtypes were differentiated on the basis of Quellungreaction using specific antisera. See, e.g., U.S. Pat. No. 5,847,112.The obtained isolates were further clonally isolated by plating seriallyin two stages on agar plates consisting of an animal-component freemedium containing soy peptone, yeast extract, and glucose without hemin.For serotype 7F, the agar plates used also contained hemin. Clonalisolates for each serotype were further expanded in liquid culture usinganimal-component free media containing soy peptone, yeast extract,HEPES, sodium chloride, sodium bicarbonate, potassium phosphate,glucose, and glycerol to prepare the pre-master cell banks.

The production of each serotype of pneumococcal polysaccharide consistedof a cell expansion and batch production fermentation followed bychemical inactivation prior to downstream purification. A thawed cellbank vial from each serotype was expanded using a shake flask or culturebottle containing a pre-sterilized animal-component free growth mediacontaining soy peptone or soy peptone ultrafiltrate, yeast extract oryeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate,potassium phosphate, and glucose. The cell expansion culture was grownin a sealed shake flask or bottle to minimize gas exchange withtemperature and agitation control. During the cell expansion of theseserotypes, temperature, pH, pressure, and agitation were controlled.Airflow overlay was also controlled as sparging was not used. Afterachieving a specified culture density, as measured by optical density at600 nm, a portion of the cell expansion culture was transferred to aproduction fermentor containing pre-sterilized animal-component freegrowth media containing soy peptone or soy peptone ultrafiltrate, yeastextract or yeast extract ultrafiltrate, sodium chloride, potassiumphosphate, and glucose. Temperature, pH, pressure, and agitation werecontrolled. Airflow overlay was also controlled as sparging was notused.

The batch fermentation was terminated via the addition of a chemicalinactivating agent, phenol, when glucose was nearly exhausted. Purephenol was added to a final concentration of 0.8-1.2% to inactivate thecells and liberate the capsular polysaccharide from the cell wall.Primary inactivation occurs for a specified time within the fermentorwhere temperature and agitation continue to be controlled. After primaryinactivation, the batch was transferred to another vessel where it washeld for an additional specified time at controlled temperature andagitation for complete inactivation. This was confirmed by eithermicrobial plating techniques or by verification of the phenolconcentration and specified time. The inactivated broth was thenpurified.

Example 2 Purification of Pneumococcal Polysaccharides

The purification process for the pneumococcal polysaccharides consistedof several centrifugation, depth filtration, concentration/diafiltrationoperations, and precipitation steps. All procedures were performed atroom temperature unless otherwise specified.

Inactivated broth from the fermentor cultures of S. pneumoniae wereflocculated with a cationic polymer (such as BPA-1000, TRETOLITE® (BakerHughes Inc., Houston, Tex.), Spectrum 8160, poly(ethyleneimine), andMillipore pDADMAC). The cationic polymers binded to the impurityproteins, nucleic acids and cell debris. Following the flocculation stepand an aging period, flocculated solids were removed via centrifugationand multiple depth filtration steps. Clarified broth was concentratedand diafiltered using a 100 kDa to 500 kDa MWCO (molecular weightcutoff) filter. Diafiltration was accomplished using Tris, MgCl₂ bufferand sodium phosphate buffer. Diafiltration removed residual nucleic acidand protein.

Removal of further impurities was accomplished by reprecipitation of thepolysaccharide in sodium acetate and phenol with denatured alcoholand/or isopropanol. During the phenol precipitation step, sodium acetatein sodium phosphate saline buffer and phenol (liquefied phenols or solidphenols) were charged to the diafiltered retentate. Alcoholfractionation of the polysaccharide was then conducted in two stages. Inthe first stage a low percent alcohol was added to the preparation toprecipitate cellular debris and other unwanted impurities, while thecrude polysaccharide remained in solution. The impurities were removedvia centrifugation followed by a depth filtration step. Thepolysaccharide was then recovered from the solution by adding additionalisopropanol or denatured alcohol to the batch. The precipitatedpolysaccharide pellet was recovered by centrifugation, triturated anddried as a powder and stored frozen at −70° C.

Example 3 Preparation of Serotype 1 Conjugate for PCV23 (DMSO)Polyvalent Study Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in dimethylsulfoxide (DMSO). Redissolved polysaccharide andCRM197 solutions were then combined and conjugated as described below.The resulting conjugate was purified by dialysis prior to a final0.2-micron filtration. Several process parameters within each step, suchas pH, temperature, concentration, and time were controlled to yieldconjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 250 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 15 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membranefollowed by diafiltration against water using a 5 kDa NMWCO tangentialflow ultrafiltration membrane. Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 2.5 mgPs/mL with sucrose concentration of 10% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was dialyzed at approximately 4° C. for 3 days against 150 mMsodium chloride, 0.05% (w/v) polysorbate 20, using a 300 kDa NMWCOdialysis casette.

Final Filtration and Product Storage

The batch was 0.2 micron filtered (with 0.5 micron prefilter), dispensedinto aliquots and frozen at ≤−60° C.

Example 4 Preparation of Serotype 1 Conjugate for PCV23 (DMSO+Aq)Polyvalent Study Using Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 250 bar/5passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 15 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 7.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was combined with thebuffered polysaccharide solution at a polysaccharide to CRM197 massratio of 0.5. The mass ratio was selected to control the polysaccharideto CRM197 ratio in the resulting conjugate. The polysaccharide andphosphate concentrations were 6.9 g/L and 100 mM respectively. Thepolysaccharide concentration was selected to control the size of theresulting conjugate. Nickel chloride was added to approximately 2 mMusing a 100 mM nickel chloride solution. Sodium cyanoborohydride (2moles per mole of polysaccharide repeating unit) was added. Conjugationproceeded for 120 hours to maximize consumption of polysaccharide andprotein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was addedto the retentate batch to a concentration of 0.05% (w/v) then the batchwas 0.2 micron filtered.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 bufferwith 0.015% (w/v) polysorbate 20. The batch was dispensed into aliquotsand frozen at ≤−60° C.

Example 5 Preparation of Serotype 1 Conjugate for PCV22 Polyvalent StudyUsing Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 250 bar/5passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 15 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 7.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was combined with thebuffered polysaccharide solution at a polysaccharide to CRM197 massratio of 0.5. The mass ratio was selected to control the polysaccharideto CRM197 ratio in the resulting conjugate. The polysaccharide andphosphate concentrations were 6.9 g/L and 100 mM respectively. Thepolysaccharide concentration was selected to control the size of theresulting conjugate. The solution was then 0.2-micron filtered. Nickelchloride was added to approximately 2 mM using a 100 mM nickel chloridesolution. Sodium cyanoborohydride (2 moles per mole of polysacchariderepeating unit) was added. Conjugation proceeded for 120 hours tomaximize consumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was addedto the retentate batch to a concentration of 0.05% (w/v) then the batchwas 0.2 micron filtered.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 bufferwith 0.015% (w/v) polysorbate 20. The batch was dispensed into aliquotsand frozen at ≤−60° C.

Example 6 Preparation of Serotype 3 Conjugate for PCV 23 (DMSO)Polyvalent Study Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in dimethylsulfoxide (DMSO). Redissolved polysaccharide andCRM197 solutions were then combined and conjugated as described below.The resulting conjugate was purified by ultrafiltration prior to a final0.2-micron filtration. Several process parameters within each step, suchas pH, temperature, concentration, and time were controlled to yieldconjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 810 bar/6 passesfollowed by 900 bar/3 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 12 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 2.5 mgPs/mL with sucrose concentration of 10% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.25 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.35. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 7 Preparation of Serotype 3 Conjugate for PCV22 and PCV23(DMSO+Aq) Polyvalent Studies Using Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 380 bar/5passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 12 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 6.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was 0.2-micron filtered andcombined with the buffered polysaccharide solution at a polysaccharideto CRM197 mass ratio of 0.6. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Thepolysaccharide and phosphate concentrations were 4.1 g/L and 150 mMrespectively. The polysaccharide concentration was selected to controlthe size of the resulting conjugate. The solution was then 0.2-micronfiltered. Nickel chloride was added to approximately 2 mM using a 100 mMnickel chloride solution. Sodium cyanoborohydride (2 moles per mole ofpolysaccharide repeating unit) was added. Conjugation proceeded for 120hours at 10° C. to maximize consumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. The batch was 0.2 micronfiltered.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer.The batch was dispensed into aliquots and frozen at ≤−60° C.

Example 8 Preparation of Serotype 4 Conjugate for PCV23 (DMSO)Polyvalent Study Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by dialysis prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 300 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 50° C. and pH 4.1 witha sodium acetate buffer to partially deketalize the polysaccharide. Thepolysaccharide solution was then cooled to 22° C. prior to activation.Polysaccharide activation was initiated with the addition of a 100 mMsodium metaperiodate solution. The oxidation reaction proceeded for 4hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membranefollowed by diafiltration against water using a 5 kDa NMWCO tangentialflow ultrafiltration membrane. Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 5.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 2.0. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was dialyzed at approximately 4° C. for 3 days against 150 mMsodium chloride, 0.05% (w/v) polysorbate 20, using a 300 kDa NMWCOdialysis cassette.

Final Filtration and Product Storage

The batch was 0.2 micron filtered (with 0.5 micron prefilter), dispensedinto aliquots and frozen at ≤−60° C.

Example 9 Preparation of Serotype 4 Conjugate for PCV23 (DMSO+Aq) andPCV22 Polyvalent Studies Using Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 300 bar/5passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 50° C. and pH 4.1 witha sodium acetate buffer to partially deketalize the polysaccharide. Thepolysaccharide solution was then cooled to 22° C. prior to activation.Polysaccharide activation was initiated with the addition of a 100 mMsodium metaperiodate solution. The oxidation reaction proceeded for 4hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 7.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was 0.2-micron filtered andcombined with the buffered polysaccharide solution at a polysaccharideto CRM197 mass ratio of 0.5. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Thepolysaccharide and phosphate concentrations were 8.3 g/L and 100 mMrespectively. The polysaccharide concentration was selected to controlthe size of the resulting conjugate. The solution was then 0.2-micronfiltered. Nickel chloride was added to approximately 2 mM using a 100 mMnickel chloride solution. Sodium cyanoborohydride (2 moles per mole ofpolysaccharide repeating unit) was added. Conjugation proceeded for 120hours to maximize consumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. Then the batch was 0.2micron filtered.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer.The batch was dispensed into aliquots and frozen at ≤−60° C.

Example 10 Preparation of Serotype 5 Conjugate for PCV23 (DMSO)Polyvalent Study Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by dialysis prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 600 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4° C. and pH 4.1 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 4 hours at 4° C.

The activated product was diafiltered against 10 mM Sodium Acetate, pH4.1 using a 10 kDa NMWCO tangential flow ultrafiltration membranefollowed by diafiltration against water using a 5 kDa NMWCO tangentialflow ultrafiltration membrane. Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a concentration of 20 mM. The polysaccharide andCRM197 solutions were blended to achieve a polysaccharide concentrationof 2.0 g Ps/L and a polysaccharide to CRM197 mass ratio of 1.5. The massratio was selected to control the polysaccharide to CRM197 ratio in theresulting conjugate. Sodium cyanoborohydride (1 mole per mole ofpolysaccharide repeating unit) was added, and conjugation proceeded at22° C.

Dilution and Neutralization

The batch was diluted into 150 mM sodium chloride, with approximately0.025% (w/v) polysorbate 20, at approximately 4° C. Potassium phosphatebuffer was then added to neutralize the pH. The batch was dialyzed atapproximately 4° C. for 3 days against 150 mM sodium chloride, 0.05%(w/v) polysorbate 20, using a 300 kDa NMWCO dialysis cassette.

Final Filtration and Product Storage

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter),dispensed into aliquots and frozen at ≤−60° C.

Example 11 Preparation of Serotype 5 Conjugate for PCV22 and PCV23(DMSO+Aq) Polyvalent Studies Using Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 600 bar/5passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4° C. and pH 4.1 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 4 hours at 4° C.

The activated product was diafiltered against 10 mM sodium acetate, pH4.1 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 6.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was combined with thebuffered polysaccharide solution at a polysaccharide to CRM197 massratio of 0.4. The mass ratio was selected to control the polysaccharideto CRM197 ratio in the resulting conjugate. The polysaccharide andphosphate concentrations were 3.8 g/L and 150 mM respectively. Thepolysaccharide concentration was selected to control the size of theresulting conjugate. The solution was then 0.2-micron filtered. Nickelchloride was added to approximately 2 mM using a 100 mM nickel chloridesolution. Sodium cyanoborohydride (2 moles per mole of polysacchariderepeating unit) was added. Conjugation proceeded for 96 hours tomaximize consumption of polysaccharide and protein.

Purification and Neutralization

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 300 mMsodium bicarbonate pH 9.3 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then neutralized with 1.5 M potassium phosphate, pH 6.0.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was addedto the retentate batch to a concentration of 0.05% (w/v) and then thebatch was 0.2 micron filtered.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 bufferwith 0.015% (w/v) polysorbate 20. The batch was dispensed into aliquotsand frozen at ≤−60° C.

Example 12 Preparation of Serotype 6A Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) and PCV 22 Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.5 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.4. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 13 Preparation of Serotype 6B Conjugate for PCV 23 (DMSO) andPCV 23 (DMSO/Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.85 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.35. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 14 Preparation of Serotype 6B Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.75 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.35. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 15 Preparation of Serotype 7F Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 150 bar/7 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 4 hours at 4° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.6 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 16 Preparation of Serotype 7F Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 150 bar/7 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 4 hours at 4° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide conjugation to CRM97

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.04 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 17

Preparation of Serotype 8 Conjugate for PCV23 (DMSO) and PCV23 (DMSO+Aq)Polyvalent Studies Using DMSO Conjugation Polysaccharide was dissolved,sized to a target molecular mass, chemically activated andbuffer-exchanged by ultrafiltration. Activated polysaccharide andpurified CRM197 were individually lyophilized and redissolved in DMSO.Redissolved polysaccharide and CRM197 solutions were then combined andconjugated as described below. The resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 600 bar/6 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 4 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 4.5 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. After the blend, the conjugation reaction proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 18 Preparation of Serotype 9V Conjugate for PCV23 (DMSO)Polyvalent Study Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 230 bar/5.5 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 6 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.3. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with a 0.5 micronprefilter) then diluted with additional 10 mM histidine in 150 mM sodiumchloride, pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed intoaliquots and frozen at ≤−60° C.

Example 19 Preparation of Serotype 9V Conjugate for PCV22 PolyvalentStudy Using Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 100 bar/5passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 6 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 7.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was 0.2-micron filtered andcombined with the buffered polysaccharide solution at a polysaccharideto CRM197 mass ratio of 0.7. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Thepolysaccharide and phosphate concentrations were 10.0 g/L and 100 mMrespectively. The polysaccharide concentration was selected to controlthe size of the resulting conjugate. The solution was then 0.2-micronfiltered. Nickel chloride was added to approximately 2 mM using a 100 mMnickel chloride solution. Sodium cyanoborohydride (2 moles per mole ofpolysaccharide repeating unit) was added. Conjugation proceeded for 120hours to maximize consumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was addedto the retentate batch to a concentration of 0.05% (w/v) then the batchwas 0.2 micron filtered.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 bufferwith 0.015% (w/v) polysorbate 20. The batch was dispensed into aliquotsand frozen at ≤−60° C.

Example 20 Preparation of Serotype 9V Conjugate for PCV23 (DMSO+Aq)Polyvalent Study Using Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 100 bar/5passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 6 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 7.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was 0.2-micron filtered andcombined with the buffered polysaccharide solution at a polysaccharideto CRM197 mass ratio of 0.7. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Thepolysaccharide and phosphate concentrations were 10.0 g/L and 100 mMrespectively. The polysaccharide concentration was selected to controlthe size of the resulting conjugate. The solution was then 0.2-micronfiltered. Nickel chloride was added to approximately 2 mM using a 100 mMnickel chloride solution. Sodium cyanoborohydride (2 moles per mole ofpolysaccharide repeating unit) was added. Conjugation proceeded for 120hours to maximize consumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was addedto the retentate batch to a concentration of 0.05% (w/v) then the batchwas 0.2 micron filtered.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 bufferwith 0.015% (w/v) polysorbate 20. The batch was dispensed into aliquotsand frozen at ≤−60° C.

Example 21 Preparation of Serotype 10A Conjugate for PCV23 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 615 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.5 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.6. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH.

Final Filtration and Product Storage

The batch was concentrated and diafiltered against 10 mM histidine in150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4°C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 22 Preparation of Serotype 10A Conjugate for PCV23 (DMSO+Aq)Polyvalent Study Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 600 bar/5 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.4 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.6. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 150 mM sodiumchloride, 25 mM potassium phosphate, pH 7 followed by diafiltrationagainst 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015%(w/v) polysorbate 20, at 4° C. using a 300 kDaNMWCO tangential flowultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 23 Preparation of Serotype 10A Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 600 bar/5 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.8 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.75. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example X Preparation of Serotype 11A Conjugate for PCV24 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 800 bar/8 passes.Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide conjugation to CRMl97

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.3 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 24 Preparation of Serotype 12F Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 80° C. for 155minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.7 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.8. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 25 Preparation of Serotype 12F Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 90° C. for 60minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7.0, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 26 Preparation of Serotype 14 Conjugate for PCV23 (DMSO)Polyvalent Study Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by dialysis prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/6 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 4 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 1.8 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was dialyzed at approximately 4° C. for 22.5 hours against 150 mMsodium chloride, 0.05% polysorbate 20, using a 300 kDa MWCO dialysiscassette.

Final Filtration and Product Storage

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter),dispensed into aliquots and frozen at ≤−60° C.

Example 27 Preparation of Serotype 14 Conjugate for PCV22 and PCV23(DMSO+Aq) Polyvalent Study Using Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 200 bar/6passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 4 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 7.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was 0.2-micron filtered andcombined with the buffered polysaccharide solution at a polysaccharideto CRM197 mass ratio of 1.0. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Thepolysaccharide and phosphate concentrations were 3.8 g/L and 100 mMrespectively. The polysaccharide concentration was selected to controlthe size of the resulting conjugate. The solution was then 0.2-micronfiltered. Nickel chloride was added to approximately 2 mM using a 100 mMnickel chloride solution. Sodium cyanoborohydride (2 moles per mole ofpolysaccharide repeating unit) was added. Conjugation proceeded for 72hours to maximize consumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. The batch was then 0.2micron filtered.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L withadditional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer.The batch was dispensed into aliquots and frozen at ≤−60° C.

Example 28 Preparation of Serotype 15A Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 210 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 20 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO which was pre-heated to 34° C. The polysaccharidesolution was spiked with sodium chloride to a concentration of 25 mM.The polysaccharide and CRM197 solutions were blended to achieve apolysaccharide concentration of 5.0 g Ps/L and a polysaccharide toCRM197 mass ratio of 2.0. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Sodiumcyanoborohydride (1 mole per mole of polysaccharide repeating unit) wasadded, and conjugation proceeded at 34° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at34° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 29 Preparation of Serotype 15A Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 200 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 20 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO which was pre-heated to 34° C. The polysaccharidesolution was spiked with sodium chloride to a concentration of 25 mM.The polysaccharide and CRM197 solutions were blended to achieve apolysaccharide concentration of 5.0 g Ps/L and a polysaccharide toCRM197 mass ratio of 2.0. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Sodiumcyanoborohydride (1 mole per mole of polysaccharide repeating unit) wasadded, and conjugation proceeded at 34° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 30 Preparation of Serotype 15C Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide derived from Streptococcus pneumoniae serotype 15B wasdissolved, sized to a target molecular mass, subjected to mild basehydrolysis to release O-acetyl groups, chemically activated andbuffer-exchanged by ultrafiltration. Activated polysaccharide andpurified CRM197 were individually lyophilized and redissolved in DMSO.Redissolved polysaccharide and CRM197 solutions were then combined andconjugated as described below. The resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction, Base Hydrolysis and Oxidation

Purified serotype 15B pneumococcal capsular Ps powder was dissolved inwater and 0.45-micron filtered. Dissolved polysaccharide was homogenizedto reduce the molecular mass of the Ps. Homogenization pressure andnumber of passes through the homogenizer were controlled to 300 bar/5passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was heated to 60° C. and sodium bicarbonatepH 9 buffer was added to a final concentration of 50 mM. The batch wasincubated with mixing for 13 hours at 60° C. to release O-acetyl groups.Potassium phosphate pH 6 buffer was added to a final concentration of136 mM to neutralize pH and the solution was cooled to ambienttemperature. The solution was then concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.75. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 31 Preparation of Serotype 15C Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide derived from Streptococcus pneumoniae serotype 15B wasdissolved, sized to a target molecular mass, subjected to mild basehydrolysis to release O-acetyl groups, chemically activated andbuffer-exchanged by ultrafiltration. Activated polysaccharide andpurified CRM197 were individually lyophilized and redissolved in DMSO.Redissolved polysaccharide and CRM197 solutions were then combined andconjugated as described below. The resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction, Base Hydrolysis and Oxidation

Purified serotype 15B pneumococcal capsular Ps powder was dissolved inwater and 0.45-micron filtered. Dissolved polysaccharide was homogenizedto reduce the molecular mass of the Ps. Homogenization pressure andnumber of passes through the homogenizer were controlled to 300 bar/5passes. The size-reduced polysaccharide solution was heated to 60° C.and sodium bicarbonate pH 9.4 buffer was added to a final concentrationof 50 mM. The batch was incubated with mixing for 12 hours at 60° C. torelease O-acetyl groups. Potassium phosphate pH 6 buffer was added to afinal concentration of 150 mM to neutralize pH and the solution wascooled to ambient temperature. The solution was then concentrated anddiafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.2 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.75. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 32 Preparation of Serotype 18C Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 90° C. for 160minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.49 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 33 Preparation of Serotype 18C Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 90° C. for 160minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.49 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 34 Preparation of Serotype 19A Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.22-micron filtered. The polysaccharide was concentrated anddiafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 20 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.8 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.33. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 35 Preparation of Serotype 19A Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.22-micron filtered. The polysaccharide was concentrated anddiafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 20 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 3.8 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.33. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 36 Preparation of Serotype 19F Conjugate for PCV23 (DMSO), PCV23(DMSO+Aq) and PCV22 Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 150 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 4 hours at 4° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.2. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane. The retentate batch was0.2 micron filtered then incubated at 22° C. for 4.5 days.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDa NMWCOtangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 37 Preparation of Serotype 22F Conjugate for PCV23 (DMSO)Polyvalent Study Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 810 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.3 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered with 0.5 micron prefilterthen diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 38 Preparation of Serotype 22F Conjugate for PCV22 and PCV23(DMSO+Aq) Polyvalent Studies Using Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 350 bar/5passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 7.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was 0.2-micron filtered andcombined with the buffered polysaccharide solution at a polysaccharideto CRM197 mass ratio of 0.6. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Thepolysaccharide and phosphate concentrations were 7.5 g/L and 100 mMrespectively. The polysaccharide concentration was selected to controlthe size of the resulting conjugate. The solution was then 0.2-micronfiltered. Nickel chloride was added to approximately 2 mM using a 100 mMnickel chloride solution. Sodium cyanoborohydride (2 moles per mole ofpolysaccharide repeating unit) was added. Conjugation proceeded for 120hours to maximize consumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane.

The batch was 0.2 micron filtered and was adjusted to a polysaccharideconcentration of 1.0 g/L with additional 10 mM L-histidine in 150 mMsodium chloride, pH 7.0 buffer. The batch was dispensed into aliquotsand frozen at ≤−60° C.

Example 39 Preparation of Serotype 23B Conjugate for PCV23 (DMSO), PCV23(DMSO+Aq) and PCV22 Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 400 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 5.0 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.5. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 40 Preparation of Serotype 23F Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 400 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 5 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.1 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.25. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 41 Preparation of Serotype 23F Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 400 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 4 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.1 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.25. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDaNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Example 42 Preparation of Serotype 24F Conjugate for PCV23 (DMSO) andPCV23(DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 80° C. for 150minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 2 mg Ps/mLwith sucrose concentration of 10% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a final concentration of 10 mM. The polysaccharideand CRM197 solutions were blended to achieve a polysaccharideconcentration of 1.4 g Ps/L and a polysaccharide to CRM197 mass ratio of1.5. The mass ratio was selected to control the polysaccharide to CRM197ratio in the resulting conjugate. Sodium cyanoborohydride (1 mole permole of polysaccharide repeating unit) was added, and conjugationproceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 43 Preparation of Serotype 24F Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was size-reduced by acidhydrolysis by adding acetic acid to 200 mM, incubating at 80° C. for 150minutes, then neutralizing by adding cold potassium phosphate pH 7buffer to 400 mM.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 2 mg Ps/mLwith sucrose concentration of 10% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a final concentration of 25 mM. The polysaccharideand CRM197 solutions were blended to achieve a polysaccharideconcentration of 1.4 g Ps/L and a polysaccharide to CRM197 mass ratio of1.5. The mass ratio was selected to control the polysaccharide to CRM197ratio in the resulting conjugate. Sodium cyanoborohydride (1 mole permole of polysaccharide repeating unit) was added, and conjugationproceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 44 Preparation of Serotype 33F Conjugate for PCV23 (DMSO)Polyvalent Study Using DMSO Conjugation

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in DMSO. Redissolved polysaccharide and CRM197 solutionswere then combined and conjugated as described below. The resultingconjugate was purified by ultrafiltration prior to a final 0.2-micronfiltration. Several process parameters within each step, such as pH,temperature, concentration, and time were controlled to yield conjugateswith desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 510 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a polysaccharide concentration of 2.5 g Ps/L and apolysaccharide to CRM197 mass ratio of 1.75. The mass ratio was selectedto control the polysaccharide to CRM197 ratio in the resultingconjugate. Sodium cyanoborohydride (1 mole per mole of polysacchariderepeating unit) was added, and conjugation proceeded at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 45 Preparation of Serotype 33F Conjugate for PCV22 and PCV23(DMSO+Aq) Polyvalent Studies Using Aqueous Conjugation

Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM197 was then conjugatedto the activated polysaccharide utilizing nickel chloride in thereaction mixture, and the resulting conjugate was purified byultrafiltration prior to a final 0.2-micron filtration. Several processparameters within each step, such as pH, temperature, concentration, andtime were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater, and 0.45-micron filtered. Dissolved polysaccharide washomogenized to reduce the molecular mass. Homogenization pressure andnumber of passes through the homogenizer were controlled to 350 bar/4passes to size-reduce to a target molecular mass. Size-reducedpolysaccharide was then concentrated and diafiltered against water usinga 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Ultrafiltration was conducted at 2-8° C.

Polysaccharide Conjugation to CRM197

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate pH 7.0. The buffer pH selected was to improve thestability of activated polysaccharide during the conjugation reaction.Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was 0.2-micron filtered andcombined with the buffered polysaccharide solution at a polysaccharideto CRM197 mass ratio of 0.7. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Thepolysaccharide and phosphate concentrations were 6.5 g/L and 100 mMrespectively. The polysaccharide concentration was selected to controlthe size of the resulting conjugate. The solution was then 0.2-micronfiltered. Nickel chloride was added to approximately 2 mM using a 100 mMnickel chloride solution. Sodium cyanoborohydride (2 moles per mole ofpolysaccharide repeating unit) was added. Conjugation proceeded for 96hours to maximize consumption of polysaccharide and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to apolysaccharide concentration of approximately 3.5 g/L, cooled to 2-8°C., and 1.2-micron filtered. The batch was diafiltered against 100 mMpotassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa NMWCO tangentialflow ultrafiltration membrane. The batch, recovered in the retentate,was then diluted to approximately 2.0 g polysaccharide/L and pH-adjustedwith the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1 mole per mole of polysaccharide repeating unit) wasadded. 1.5 M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mML-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDaNMWCO tangential flow ultrafiltration membrane. The batch was 0.2 micronfiltered and was adjusted to a polysaccharide concentration of 1.0 g/Lwith additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0buffer. The batch was dispensed into aliquots and frozen at ≤−60° C.

Example 46 Preparation of Serotype 35B Conjugate for PCV23 (DMSO) andPCV23 (DMSO+Aq) Polyvalent Studies Using DMSO Conjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was concentrated anddiafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a final concentration of 20 mM. The polysaccharideand CRM197 solutions were blended to achieve a polysaccharideconcentration of 6.0 g Ps/L and a polysaccharide to CRM197 mass ratio of3.0. The mass ratio was selected to control the polysaccharide to CRM197ratio in the resulting conjugate. Conjugation proceeded at 34° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at34° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example 47 Preparation of Serotype 35B Conjugate for PCV22 PolyvalentStudy Using DMSO Conjugation

Polysaccharide was dissolved, chemically activated and buffer-exchangedby ultrafiltration. Activated polysaccharide and purified CRM197 wereindividually lyophilized and redissolved in DMSO. Redissolvedpolysaccharide and CRM197 solutions were then combined and conjugated asdescribed below. The resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to yield conjugates with desired attributes.

Polysaccharide Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was concentrated anddiafiltered against water using a 10 kDa NMWCO tangential flowultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The oxidation reaction proceededfor 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharide was formulated for lyophilization at 6 mg Ps/mLwith sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a final concentration of 20 mM. The polysaccharideand CRM197 solutions were blended to achieve a polysaccharideconcentration of 6.0 g Ps/L and a polysaccharide to CRM197 mass ratio of3.0. The mass ratio was selected to control the polysaccharide to CRM197ratio in the resulting conjugate. Conjugation proceeded at 34° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 moles per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at34° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron prefilter)then diluted with additional 10 mM histidine in 150 mM sodium chloride,pH 7.0 with 0.015% (w/v) polysorbate 20, dispensed into aliquots andfrozen at ≤−60° C.

Example Y Preparation of Serotypes for PCV24 Polyvalent Study Using DMSOConjugation

Conjugates were prepared for the PCV24 study using methods similar tothose described in prior Examples. For each serotype, polysaccharide wasdissolved, chemically activated and buffer-exchanged by ultrafiltration.Activated polysaccharide and purified CRM197 were individuallylyophilized and redissolved in DMSO. Redissolved polysaccharide andCRM197 solutions were then combined and conjugated as described below.The resulting conjugate was purified by ultrafiltration prior to a final0.2-micron filtration.

Several process parameters within each step, such as pH, temperature,concentration, and time were controlled to yield conjugates with desiredattributes. Differences from prior Examples may include: homogenizationpressure and number of passes, oxidation time, polysaccharide andsucrose concentration for lyophilization, polysaccharide concentrationduring conjugation, polysaccharide to CRM197 mass ratio and saltconcentration in the conjugation reaction.

Example 48 Formulation of Pneumococcal Conjugate Vaccines

Individual pneumococcal polysaccharide-protein conjugates preparedutilizing different chemistries as described in the Examples, supra,were used for the formulation of an 8-, 15-, 22-, 23- & 24-valentpneumococcal conjugate vaccines.

The PCV8/APA vaccine drug product is prepared by individuallyconjugating the CRM197 protein to Pneumococcal polysaccharide (PnPs)Types (-8, -10A, -12F, -15A, -15C, -23B, -24F and -35B) using reductiveamination in an aprotic solvent (also referred to as DMSO chemistry) andformulated in 20 mM L-Histidine pH 5.8 and 150 mM NaCl 0.1% w/vPolysorbate-20 (PS-20) and 250 μg [Al]/mL in the form of AluminumPhosphate Adjuvant as the adjuvant at 4 μg/mL each serotype for a totalpolysaccharide concentration of 32 μg/mL.

The PCV15/APA vaccine drug product is prepared by individuallyconjugating the CRM197 protein to Pneumococcal polysaccharide (PnPs)Types (-6A, -6B, -7F, -9V, -18C, -19A, -19F, -23F) using reductiveamination in an aprotic solvent (also referred to as DMSO chemistry) orfor Types -1, -3, -4, -5, -14, -22F and -33F using reductive aminationin a protic solvent (also referred to as aqueous chemistry) andformulated in 20 mM L-Histidine pH 5.8 and 150 mM NaCl 0.2% w/vPolysorbate-20 (PS-20) and 250 μg [Al]/mL in the form of AluminumPhosphate as the adjuvant at 4 μg/mL each serotype (except 6B at 8μg/mL) for a total polysaccharide concentration of 64 μg/mL.

The PCV22 vaccine drug product used to immunize mice and rabbits wasprepared by individually conjugating the CRM197 protein to Pneumococcalpolysaccharide (PnPs) Types (-1, -3, -4, -5, -6A, -6B, -7F, -9V, -10A,-12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F,and -35B) using reductive amination in a protic and aprotic solutions(DMSO/Aqueous “Aq”) and formulated in 20 mM L-Histidine pH 5.8 and 150mM NaCl and 0.2% w/v Polysorbate-20 (PS-20) at 0.8 μg/mL each serotype(except 6B at 1.6 g/mL) for a total polysaccharide concentration of 18.4μg/mL referred to as PCV22 unadjuvanted or unadj. In another specificembodiment, the formulation is prepared with 50 μg [Al]/mL in the formof Aluminum Phosphate as the adjuvant referred to as PCV22/APA.

The PCV23 vaccine drug product is prepared by individually conjugatingthe CRM197 protein to Pneumococcal polysaccharide (PnPs) Types (-1, -3,-4, -5, -6A, -6B, -7F, -8, -9V, -10A, -12F, -14, -15A, -15C, -18C, -19A,-19F, -22F, -23B, -23F, -24F, -33F, and -35B) using reductive aminationin an aprotic solvent (also referred to as DMSO chemistry) andformulated in 20 mM L-Histidine pH 5.8 and 150 mM NaCl and 0.2% w/vPolysorbate-20 (PS-20) at 4 μg/mL each -serotype for a totalpolysaccharide concentration of 92 μg/mL referred to as PCV23 unadjuv.In another specific embodiment, the formulation is prepared with 250 μg[Al]/mL in the form of Aluminum Phosphate Adjuvant as the adjuvantreferred to as PCV23/APA. In another embodiment, the PCV23 vaccine drugproduct is prepared by individually conjugating the CRM197 protein toPneumococcal polysaccharide (PnPs) Types (-1, -3, -4, -5, -9V, -14,-22F, and -33F) using reductive amination in an protic solvent (alsoreferred to as aqueous chemistry) and conjugating the CRM197 protein toPneumococcal polysaccharide (PnPs) Types (-6A, -6B, -7F, -8, -10A, -12F,-15A, -15C, -18C, -19A, -19F, -23B, -23F, -24F, and -35B) usingreductive amination in an aprotic solvent (also referred to as DMSOchemistry). The vaccine drug product is formulated in 20 mM L-HistidinepH 5.8 and 150 mM NaCl and 0.1% w/v Polysorbate-20 (PS-20) with 0.01%CarboxyMethyl Cellulose (CMC) at 4 μg/mL each serotype (except 6B at 8μg/mL) for a total polysaccharide concentration of 96 μg/mL and preparedwith 250 μg [Al]/mL in the form of Aluminum Phosphate Adjuvant as theadjuvant and referred to as PCV23 (DMSO+Aq)/APA.

The multivalent immunogenic composition PCV24 is prepared byindividually conjugating the CRM197 protein to S. pneumoniaepolysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8, -9V,-10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F,-24F, -33F, and -35B using reductive amination in an aprotic solvent(also referred to as DMSO chemistry) and formulated in 20 mM L-HistidinepH 5.8, 150 mM NaCl and 0.1% w/v Polysorbate-20 (PS-20) at 4 μg/mL or 8μg/mL of each polysacchardide serotype for a total polysaccharideconcentration of 96 μg/mL or 192 μg/mL, respectively, and referred to as“PCV24 unadj”. In another specific embodiment, the multivalentimmunogenic composition PCV24 is prepared in 20 mM L-Histidine pH 5.8,150 mM NaCl and 0.2% w/v Polysorbate-20 (PS-20) at 4 μg/mL of eachpolysaccharide serotype for a total polysaccharide concentration of 96μg/mL further comprising 250 μg [Al]/mL in the form of AluminumPhosphate Adjuvant. This is referred to as “PCV24/APA”.

The required volume of bulk conjugates needed to obtain the targetconcentration of individual serotypes were calculated based on batchvolume and concentration of individual bulk polysaccharideconcentrations. The individual conjugates were added to a solution ofhistidine, sodium chloride and Polysorbate-20 (PS-20) to produce a2×-4×conjugate blend. The formulation vessel containing the conjugateblend is mixed using a magnetic stir bar, sterile filtered into anothervessel. The sterile filtered 2×-4×blend is either added to anothervessel containing Aluminum Phosphate Adjuvant or diluted with saline toachieve the desired target total polysaccharide, excipient and APAadjuvant (if required) concentrations. The formulations are then filledinto glass vials or syringes and stored at 2-8° C.

Example 49 PCV22 Immunogenicity and Functional Antibody in Mice

Young female Balb/c mice (6-8 weeks old, n=10/group) were immunized with0.1 mL of a 22-valent pneumococcal conjugate vaccine (PCV22/APA or PCV22unadjuvanted) on day 0, day 14 and day 28. PCV22 was dosed at 0.08 μg ofeach pneumococcal polysaccharide (1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14,15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B) and 6B at 0.16 μgand all conjugated to CRM197 unadjuvanted or with 5 μg aluminumphosphate adjuvant (APA) per immunization. Mice were observed at leastdaily by trained animal care staff for any signs of illness or distress.The vaccine formulations in mice were deemed to be safe and welltolerated, as no vaccine-related adverse events were noted. On day 52the mice were intratracheally challenged with Streptococcus pneumoniaeserotype 24F. Exponential phase cultures of S. pneumoniae werecentrifuged, washed, and suspended in sterile PBS. Mice wereanesthetized with isoflurane prior to challenge. 10⁵ cfu of S.pneumoniae in 0.1 mL of PBS was placed in the throat of mice hungupright by their incisors. Aspiration of the bacteria was induced bygently pulling the tongue outward and covering the nostrils. Mice wereweighed daily and euthanized if weight loss exceeded 20% of startingweight. Blood was collected at 24 hours, 48 hours and 72 hours to assessfor bacteremia. Mice were observed at least twice daily by trainedanimal care staff for any signs of illness or distress. All animalexperiments were performed in strict accordance with the recommendationsin the Guide for Care and Use of Laboratory Animals of the NationalInstitutes of Health. The mouse experimental protocol was approved bythe Institutional Animal Care and Use Committee at Merck & Co., Inc.

Mouse sera were evaluated for IgG immunogenicity using a multiplexedelectrochemiluminescence (ECL) assay. This assay was developed for usewith mouse serum based on the human assay described by Marchese etal.^([3]) using technology developed by MesoScale Discovery (a divisionof MesoScale Diagnostics, LLC, Gaithersburg, Md.) which utilizes aSULFO-TAG™ label that emits light upon electrochemical stimulation.SULFO-TAG™-labeled anti-mouse IgG was used as the secondary antibody fortesting mouse serum samples. Functional antibody was determined throughmultiplexed opsonophagocytic assays (MOPA) based on previously describedprotocols at www.vaccine.uab.edu and Opsotiter® 3 software owned by andlicensed from University of Alabama (UAB) Research Foundation ^([1, 2])Mouse sera were pooled for each group and tested in multiplexedelectrochemiluminescent assays to determine antibody titers. PCV22generated antibody titers in mice for all serotypes following 1, 2 and 3immunizations with the vaccine (FIG. 1). PCV22 showed cross-reactivityto serotype 15B, as evidenced by IgG titers (FIG. 1). PCV22 formulatedwith APA trended toward higher immunogenicity compared to unadjuvantedPCV22 in mice at PD2 (data not shown) and PD3 (FIG. 2).

Mouse sera were pooled for each group and tested in multiplexedopsonophagocytic assays (MOPA) to determine functional antibody titers.PCV22 generated functional antibody titers in mice which killedvaccine-type bacterial serotypes following 3 immunizations with thevaccine (FIG. 3). PCV22 formulated with APA trended toward higherfunctional antibody titers compared to PCV22 unadjuvanted at PD2 (datanot shown) and PD3 (FIG. 4), similar to IgG titers (FIG. 2).

PCV22 immunized mice were protected from intratracheal challenge with S.pneumoniae 24F (FIG. 5). Mantel Cox log-rank test indicated that boththe PCV22 unadjuvanted (PCV22 unadj) and PCV22/APA groups weresignificantly protected from challenge when compared to the naïve group(P<0.0001). Likewise, both PCV22 immunized mouse groups had little to nobacteremia, which was significantly less when compared to the naivegroup (data not shown).

Example 50 PCV22 Immunogenicity and Functional Antibody in Rabbits

Adult New Zealand white rabbits (NZWR, n=5/group) were intramuscularly(IM) immunized with 0.1 mL of a 22-valent pneumococcal conjugate vaccine(PCV22/APA or PCV22 unadjuvanted) on day 0 and day 14 (alternatingsides). PCV22 was dosed at 0.08 μg of each pneumococcal polysaccharide(1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,23B, 23F, 24F, 33F, 35B) and 6B at 0.16 μg and all conjugated to CRM197and either unadjuvanted or formulated with 5 μg APA per immunization.Sera were collected prior to study start (pre-immune) and on days 14(PD1) and 28 (PD2). NZWRs were observed at least daily by trained animalcare staff for any signs of illness or distress. The vaccineformulations in NZWRs were deemed to be safe and well tolerated, as novaccine-related adverse events were noted. All animal experiments wereperformed in strict accordance with the recommendations in the Guide forCare and Use of Laboratory Animals of the National Institutes of Health.The NZWR experimental protocol was approved by the Institutional AnimalCare and Use Committees at both Merck & Co., Inc and Covance (Denver,Pa.).

Rabbit sera were evaluated for IgG immunogenicity using a multiplexedelectrochemiluminescence (ECL) assay. This assay was developed for usewith rabbit serum based on the human assay described by Marchese etal.^([3]) using technology developed by MesoScale Discovery (a divisionof MesoScale Diagnostics, LLC, Gaithersburg, Md.) which utilizes aSULFO-TAG™ label that emits light upon electrochemical stimulation.SULFO-TAG™-labeled anti-rabbit IgG was used as the secondary antibodyfor testing NZWR serum samples. Functional antibody was determinedthrough multiplexed opsonophagocytic assays (MOPA) based on previouslydescribed protocols at www.vaccine.uab.edu and Opsotiter® software ownedby and licensed from University of Alabama (UAB) ResearchFoundation^([1, 2]).

Rabbit sera were tested individually in multiplexedelectrochemiluminescent assays to determine antibody titers. PCV22generated antibody titers in rabbits for all serotypes followingimmunizations with the vaccine (FIG. 6). Immunization of rabbits withPCV22 also generates antibodies that bind to serotype 15B polysaccharide(FIG. 6). There was no benefit to including APA with PCV22 in rabbits,as the immunogenicity was comparable to or lower than (serotype 1) PCV22unadjuvanted at PD2 (FIG. 7).

Rabbit sera were tested individually in multiplexed opsonophagocyticassays (MOPA) to determine functional antibody titers. PCV22 generatedfunctional antibody titers in rabbits which killed vaccine-typebacterial serotypes following 2 immunizations with the vaccine (FIG. 8).PCV22 unadjuvanted had higher functional antibody titers at PD1 forserotype 4 (data not shown) and at PD2 for serotypes 1, 3 and 4 comparedto PCV22 formulated with APA. PCV22 formulated with APA did not havehigher functional antibody titers at PD1 (data not shown) and PD2 formost of the serotypes compared to PCV22 unadjuvanted (FIG. 8).

Example 51 PCV23 Immunogenicity in Infant Rhesus Macaques

Infant Rhesus macaques (IRM, 2-3 months old, n=8-9/group) wereintramuscularly immunized with 0.1 mL of a 23-valent pneumococcalconjugate vaccine (PCV23) on days 0, 28 and 56. PCV23 was dosed at 9.6μg of total pneumococcal polysaccharide (1, 3, 4, 5, 6A, 7F, 8, 9V, 10A,12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B at 0.4μg, 6B at 0.8 μg and all conjugated to CRM197) unadjuvanted orformulated with 25 μg aluminum phosphate adjuvant (APA) perimmunization. An additional group of IRMs were intramuscularly immunizedwith a 0.1 mL of PCV15. PCV15 was dosed at 6.4 μg of total pneumococcalpolysaccharide (1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and33F at 0.4 μg, 6B at 0.8 μg and all conjugated to CRM197 with 25 μg APAper immunization) in one quadricep and 0.1 mL of PCV8 (8, 10A, 12F, 15A,15C, 23B, 24F and 35B at 0.4 μg and all conjugated to CRM197 with 25 μgAPA per immunization) in a separate quadricep following the sameschedule as described above. Sera were collected prior to study start(pre-immune, day 0) and on days 14 (PD1), 28, 42 (PD2), 56, and 70(PD3). IRMs were observed at least daily by trained animal care stafffor any signs of illness or distress. All animal experiments wereperformed in strict accordance with the recommendations in the Guide forCare and Use of Laboratory Animals of the National Institutes of Health.The experimental protocol was approved by the Institutional Animal Careand Use Committee at Merck & Co., Inc and New Iberia Research Center.

Rhesus sera were evaluated for IgG immunogenicity using a multiplexedelectrochemiluminescence (ECL) assay. This assay was developed for usewith Rhesus serum based on the human assay described by Marchese et al.and Skinner et al^([3, 4]) using technology developed by MesoScaleDiscovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, Md.)which utilizes a SULFO-TAG™ label that emits light upon electrochemicalstimulation. SULFO-TAG™-labeled anti-human IgG was used as the secondaryantibody for testing Rhesus serum samples.

IRM immunization with PCV23 generated antibody titers for all serotypesfor all of the PCV23 vaccine formulations evaluated (FIGS. 9A-9D). It isalso of note that PCV23, which contains polysaccharide conjugates15A-CRM197 and 15C-CRM197, also provides cross-reactivity to 15B, asevidenced in ECL (FIGS. 9A-9D). PCV23 was immunogenic with one dose ofvaccine in the IRMs (FIGS. 9A-9D).

The PCV23 (DMSO)/APA formulation had higher immunogenicity for serotype22F compared to PCV23 unadjuvanted at PD1 while PCV23 (DMSO+Aq)/APA hadhigher immunogenicity for serotype 1 and serotype 15C compared to PCV23unadjuvanted at PD1 (FIG. 10A). The PCV23 (DMSO)/APA formulation hadhigher immunogenicity for serotype 18C compared to PCV23 unadjuvanted atPD2 (FIG. 10B). The PCV23 (DMSO+Aq)/APA had higher immunogenicity forthe majority of serotypes (1, 3, 4, 7F, 8, 9V, 12F, 14, 15A, 15B, 15C,18C, 19F, 22F and 23F) compared to PCV23 unadjuvanted at PD2 (FIG. 10B).The PCV23 (DMSO+Aq)/APA had higher immunogenicity for serotypes 1, 4 and9V compared to PCV23 unadjuvanted at PD3 (FIG. 10C).

IRMs vaccinated with PCV23 (DMSO+Aq)/APA or with a co-administration ofPCV15/APA+PCV8/APA in separate limbs did not show many immunogenicitydifferences at PD1, with the exception of serotype 10A which had higherimmunogenicity in the co-administration group (FIG. 11A). However, IRMsvaccinated with PCV23 (DMSO+Aq)/APA had higher PD2 immunogenicity forthe majority of serotypes 1, 3, 4, 5, 7F, 8, 9V, 14, 15A, 15B, 15C, 18C,19F, 22F, 23B and 23F compared to co-administration ofPCV15/APA+PCV8/APA (FIG. 11B). There were no differences inimmunogenicity between the two vaccines at PD3 (FIG. 11C).

When evaluating the antibody boosting effects, all of the PCVs generatedprimary immune responses at PD1 compared to pre-immune sera for allserotypes with the exception of serotype 23B in IRMs immunized withPCV23 (DMSO+Aq)/APA (FIG. 12A). All PCVs evaluated generatedsignificantly higher antibody titers at PD2 and PD3 when compared topre-immune sera for all serotypes (FIGS. 12B and 12C). A second dose ofPCV results in increased antibody titers from PD2 compared to PD1 forall serotypes except for serotype 3 and serotype 1 in IRMs immunizedwith PCV23 unadjuvanted, PCV23 (DMSO)/APA and PCV15/APA+PCV8/APA (FIG.12D). A third dose of PCV results a boost in antibody titers for themajority of serotypes, with the exception of IRMs immunized with PCV23(DMSO+Aq)/APA where there is a decrease at PD3 compared to PD2,suggesting that PCV23 (DMSO+Aq)/APA immunized IRMs reached the maximumantibody response at PD2 (FIG. 12E).

Example Z PCV24 Immunogenicity in New Zealand White Rabbits and InfantRhesus Macaques

New Zealand white rabbits (NZWR, n=8/group) were intramuscularlyimmunized with 0.1 mL of a 24-valent pneumococcal conjugate vaccine(PCV24) on days 0 and 14. PCV24 was dosed at 9.6 μg of totalpneumococcal polysaccharide (1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A,12F, 14, 15A, deOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35Bat 0.4 μg and all conjugated to CRM197) and formulated with aluminumphosphate adjuvant (APA, 25 μg) per immunization. Sera were collectedprior to study start (pre-immune, day 0) and on days 14 (PD1) and 28(PD2). NZWRs were observed at least daily by trained animal care stafffor any signs of illness or distress.

Infant Rhesus macaques (IRM, 2-3 months old, n=5/group) wereintramuscularly immunized with 0.1 mL of a 24-valent pneumococcalconjugate vaccine (PCV24) on days 0, 28 and 56. PCV24 was dosed at 9.6μg of total pneumococcal polysaccharide (1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, deOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33Fand 35B at 0.4 μg and all conjugated to CRM197) and formulated withaluminum phosphate adjuvant (APA, 25 μg) per immunization. Sera werecollected prior to study start (pre-immune, day 0) and on days 14 (PD1),28, 42 (PD2), 56, and 70 (PD3). IRMs were observed at least daily bytrained animal care staff for any signs of illness or distress. Allanimal experiments were performed in strict accordance with therecommendations in the Guide for Care and Use of Laboratory Animals ofthe National Institutes of Health. The experimental protocol wasapproved by the Institutional Animal Care and Use Committee at Merck &Co., Inc and New Iberia Research Center.

Rhesus sera were evaluated for IgG immunogenicity using a multiplexedelectrochemiluminescence (ECL) assay. This assay was developed for usewith Rhesus serum based on the human assay described by Marchese et al.and Skinner et al [3, 4] using technology developed by MesoScaleDiscovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, Md.)which utilizes a SULFO-TAG™ label that emits light upon electrochemicalstimulation. SULFO-TAG™-labeled anti-human IgG was used as the secondaryantibody for testing Rhesus serum samples and a SULFO-TAG™-labeledanti-rabbit IgG for the New Zealand white rabbit samples.

Functional antibody was determined through multiplexed opsonophagocyticassays (MOPA) based on previously described protocols atwww.vaccine.uab.edu and Opsotiter® 3 software owned by and licensed fromUniversity of Alabama (UAB) Research Foundation [1, 2].

NZWR immunization with PCV24 generated antibody titers for all serotypesin the vaccine (FIG. 13A). It is also of note that PCV24, which containspolysaccharide conjugates 15A-CRM197, deOAc15B-CRM197, 6A-CRM197,6B-CRM197 also provides cross-reactivity to 15B and 6C, as evidenced inECL (FIG. 13A).

NZWR sera were tested individually in multiplexed opsonophagocyticassays (MOPA) to determine functional antibody titers. PCV24 generatedfunctional antibody titers in NZWRs which killed all vaccine-typebacterial serotypes (FIG. 13B).

IRM immunization with PCV24 generated antibody titers for all serotypesin the vaccine (FIG. 14A). It is also of note that PCV24, which containspolysaccharide conjugates 15A-CRM197, deOAc15B-CRM197, 6A-CRM197,6B-CRM197 also provides cross-reactivity to 15B and 6C, as evidenced inECL (FIG. 14A).

IRM sera were tested individually in multiplexed opsonophagocytic assays(MOPA) to determine functional antibody titers. PCV24 generatedfunctional antibody titers in IRMs which killed vaccine-type bacterialserotypes (FIG. 14B), with the exception of 33F, which also had lowerPD3/Pre binding antibody titers in ECL. However, when PCV24/APA wasevaluated in New Zealand white rabbits, PD2 33F OPA titers were over58-fold higher than pre-immune titers (FIG. 13B).

REFERENCES

-   1. Caro-Aguilar I, Indrawati L, Kaufhold R M, Gaunt C, Zhang Y,    Nawrocki D K, et al. Immunogenicity differences of a 15-valent    pneumococcal polysaccharide conjugate vaccine (PCV15) based on    vaccine dose, route of immunization and mouse strain. Vaccine 2017    Feb. 7; 35(6):865-72.-   2. Burton R L, Nahm M H. Development and validation of a fourfold    multiplexed opsonization assay (MOPA4) for pneumococcal antibodies.    Clin Vaccine Immunol 2006 September; 13(9): 1004-9.-   3. Marchese R D, Puchalski D, Miller P, Antonello J, Hammond O,    Green T, Rubinstein L J, Caulfield M J, Sikkema D. Optimization and    validation of a multiplex, electrochemiluminescence-based detection    assay for the quantitation of immunoglobulin G serotype-specific    antipneumococcal antibodies in human serum. Clin Vaccine Immunol.    2009 March; 16(3):387-96.-   4. Skinner, J. M., et al., Pre-clinical evaluation of a 15-valent    pneumococcal conjugate vaccine (PCV15-CRM197) in an infant-rhesus    monkey immunogenicity model. Vaccine, 2011. 29(48): p. 8870-8876.

Example 52 Materials and Methods Free Polysaccharide Testing

Free polysaccharide (polysaccharide that is not conjugated with CRM197)in conjugate sample is measured by first precipitating free protein andconjugates with deoxycholate (DOC) and hydrochloric acid. Precipitatesare then filtered out and the filtrates are analyzed for freepolysaccharide concentration by HPSEC/UV/MALS/RI. Free polysaccharide iscalculated as a percentage of total polysaccharide measured byHPSEC/UV/MALS/RI.

Free Protein Testing

Free polysaccharide, polysaccharide-CRM197 conjugate, and free CRM197 inconjugate samples are separated by capillary electrophoresis in micellarelectrokinetic chromatography (MEKC) mode. Briefly, samples are mixedwith MEKC running buffer containing 25 mM borate, 100 mM SDS, pH 9.3,and are separated in a preconditioned bare-fused silica capillary.Separation is monitored at 200 nm and free CRM197 is quantified with aCRM197 standard curve. Free protein results are reported as a percentageof total protein content determined by the HPSEC/UV/MALS/RI procedure.

Molecular Weight and Concentration Analysis of Conjugates UsingHPSEC/UV/MALS/RI Assay

Conjugate samples were injected and separated by high performancesize-exclusion chromatography (HPSEC). Detection was accomplished withultraviolet (UV), multi-angle light scattering (MALS) and refractiveindex (RI) detectors in series. Protein concentration was calculatedfrom UV280 using an extinction coefficient. Polysaccharide concentrationwas deconvoluted from the RI signal (contributed by both protein andpolysaccharide) using the dn/dc factors which are the change in asolution's refractive index with a change in the solute concentrationreported in mL/g. Average molecular weight of the samples werecalculated by Astra software (Wyatt Technology Corporation, SantaBarbara, Calif.) using the measured concentration and light scatteringinformation across the entire sample peak. There are multiple form ofaverage values of molecular weight for polydispersed molecules. Forexample number-average molecular weight Mn, weight-average molecularweight Mw, and z-average molecular weight Mz (Molecules, 2015, 20,10313-10341). Unless specified, the molecular weights are weight-averagemolecular weight.

Determination of Lysine Consumption in Conjugated Protein as a Measureof the Number of Covalent Attachments Between Polysaccharide and CarrierProtein

The Waters AccQ-Tag amino acid analysis (AAA) was used to measure theextent of conjugation in conjugate samples. Samples were hydrolyzedusing vapor phase acid hydrolysis in the Eldex workstation, to break thecarrier proteins down into their component amino acids. The free aminoacids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC). The derivatized samples were then analyzed using UPLCwith UV detection on a C18 column. The average protein concentration wasobtained using representative amino acids other than lysine. Lysineconsumption during conjugation (i.e., lysine loss) was determined by thedifference between the average measured amount of lysine in theconjugate and the expected amount of lysine in the starting protein.

What is claimed is:
 1. A multivalent immunogenic composition comprisingS. pneumoniae polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, and wherein the polysaccharide proteinconjugates include polysaccharides of a group of S. pneumoniae serotypesselected from the group consisting of: a) 1, 3, 4, 5, 6A, 6B, 7F, 9V,10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B; b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,19F, 22F, 23B, 23F, 24F, 33F and 35B; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,19F, 22F, 23B, 23F, 24F, 33F and 35B; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; f) 1,3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A,19F, 22F, 23B, 23F, 24F, 33F and 35B; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B; h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C,19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; i) 1, 3, 4, 5, 6A, 6B, 7F, 8,9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B; j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F and 35B; k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F,14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; l) 1, 3,4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,24F, 33F and 35B; m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C,18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n) 1, 3, 4, 5, 6B, 7F,8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33Fand 35B; o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A,19F, 22F, 23B, 23F, 24F, 33F and 35B; p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A,11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A,19F, 22F, 23B, 23F, 24F, 33F and 35B; r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A,11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,23B, 23F, 24F, 33F and 35B; t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14,15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; u) 1, 3, 4, 5,6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F,33F and 35B; v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C,19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; w) 1, 3, 4, 5, 6B, 7F, 8, 9V,10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F and 35B; y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A,12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z) 1,3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F,23B, 23F, 24F, 33F and 35B; aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A,12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; bb)1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, deO-15B, 18C, 19A, 19F, 22F,23B, 23F, 24F, 33F, 35B and 39; cc) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14,15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; dd) 1, 3,4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, deO-15B, 18C, 19A, 19F, 22F, 23B,23F, 24F, 33F, 35B and 39; ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14,15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ff) 1, 3,4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F,24F, 33F, 35B and 39; gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14,15A, deO-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; hh) 1,3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,23B, 23F, 24F, 33F, 35B and 39; ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A,12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F, 35B and 39; kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F,14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ll) 1,3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F,24F, 33F, 35B and 39; mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C,18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; nn) 1, 3, 4, 5, 6A,7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F,35B and 39; oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A,19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; pp) 1, 3, 4, 5, 6C, 7F, 8, 9V,12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;qq) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F, 35B and 39; rr) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A,12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F, 35B and 39; tt) 1, 3, 4, 5, 6A, 7F, 9V, 12F,14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; uu) 1,3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F,24F, 33F, 35B and 39; vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B,18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ww) 1, 3, 4, 5, 6A,7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F,35B and 39; xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A,19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; yy) 1, 3, 4, 5, 6C, 7F, 8, 9V,12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;zz) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F,22F, 23B, 23F, 24F, 33F, 35B and 39; aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V,11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and39; and bbb) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C,19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and
 39. 2. The multivalentimmunogenic composition of claim 1, wherein the immunogenic compositiondoes not comprise polysaccharide protein conjugates havingpolysaccharides from any further S. pneumoniae serotypes.
 3. Themultivalent immunogenic composition of claim 1, wherein at least one ofthe polysaccharide protein conjugates is formed by a conjugationreaction comprising an aprotic solvent.
 4. The multivalent immunogeniccomposition of claim 1, wherein each of the polysaccharide proteinconjugates is formed by a conjugation reaction comprising an aproticsolvent.
 5. The multivalent immunogenic composition of claim 3 or 4,wherein the aprotic solvent is dimethylsulfoxide (DMSO).
 6. Themultivalent immunogenic composition of claim 1, wherein the carrierprotein is selected from the group consisting of Outer Membrane ProteinComplex (OMPC), tetanus toxoid, diphtheria toxoid, protein D and CRM197.7. The multivalent immunogenic composition of claim 6, wherein thecarrier protein is CRM197.
 8. The multivalent immunogenic composition ofclaim 1, wherein the composition further comprises an adjuvant.
 9. Themultivalent immunogenic composition of claim 1, wherein the compositiondoes not comprise an adjuvant.
 10. A method for inducing an immuneresponse in a human patient comprising administering the multivalentimmunogenic composition of claim 1 to a patient.
 11. A method forinducing a protective immune response in a human patient comprisingadministering the multivalent immunogenic composition of claim 1 to apatient.
 12. A method for inducing a protective immune response againstS. pneumoniae in a human patient comprising administering themultivalent immunogenic composition of claim 1 to a patient.
 13. Themethod of any of claims 10-12, wherein the patient was previouslytreated with a multivalent pneumococcal vaccine.
 14. The method of anyof claims 10 to 13, wherein the patient is 6 weeks through 17 years ofage.
 15. A method for the prevention of pneumococcal pneumonia and/orinvasive pneumococcal disease in patients 6 weeks to 17 years of agecomprising administering the multivalent immunogenic composition ofclaim 1 to patients 6 weeks to 17 years of age.
 16. A multivalentimmunogenic composition comprising 23 distinct S. pneumoniaepolysaccharide protein conjugates, wherein each of the conjugatescomprises a capsular polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, wherein each distinct polysaccharideprotein conjugate comprises a polysaccharide from S. pneumoniaeserotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C,19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein thecarrier protein is CRM197.
 17. The multivalent immunogenic compositionof claim 16, wherein the immunogenic composition does not comprisepolysaccharide protein conjugates having polysaccharides from anyfurther S. pneumoniae serotypes.
 18. The multivalent immunogeniccomposition of claim 16, wherein each of the polysaccharide proteinconjugates is formed by a conjugation reaction comprising an aproticsolvent, wherein the aprotic solvent is dimethylsulfoxide (DMSO). 19.The multivalent immunogenic composition of claim 16, wherein thecomposition comprises an adjuvant.
 20. A multivalent immunogeniccomposition comprising 24 distinct S. pneumoniae polysaccharide proteinconjugates, wherein each of the conjugates comprises a capsularpolysaccharide from a S. pneumoniae serotype conjugated to a carrierprotein, wherein each distinct polysaccharide protein conjugatecomprises a polysaccharide from S. pneumoniae serotypes 1, 3, 4, 5, 6A,6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,23F, 24F, 33F and 35B, respectively, and wherein the carrier protein isCRM197.
 21. The multivalent immunogenic composition of claim 20, whereinthe immunogenic composition does not comprise polysaccharide proteinconjugates having polysaccharides from any further S. pneumoniaeserotypes.
 22. The multivalent immunogenic composition of claim 20,wherein each of the polysaccharide protein conjugates is formed by aconjugation reaction comprising an aprotic solvent, wherein the aproticsolvent is dimethylsulfoxide (DMSO).
 23. The multivalent immunogeniccomposition of claim 20, wherein the composition comprises an adjuvant.24. A multivalent immunogenic composition comprising 24 distinct S.pneumoniae polysaccharide protein conjugates, wherein each of theconjugates comprises a capsular polysaccharide from a S. pneumoniaeserotype conjugated to a carrier protein, wherein each distinctpolysaccharide protein conjugate comprises a polysaccharide from S.pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14,15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively,and wherein the carrier protein is CRM197.
 25. The multivalentimmunogenic composition of claim 24, wherein the immunogenic compositiondoes not comprise polysaccharide protein conjugates havingpolysaccharides from any further S. pneumoniae serotypes.
 26. Themultivalent immunogenic composition of claim 24, wherein each of thepolysaccharide protein conjugates is formed by a conjugation reactioncomprising an aprotic solvent, wherein the aprotic solvent isdimethylsulfoxide (DMSO).
 27. The multivalent immunogenic composition ofclaim 24, wherein the composition comprises an adjuvant.
 28. Amultivalent immunogenic composition comprising up to 30 distinct S.pneumoniae polysaccharide protein conjugates, wherein each of theconjugates comprises a polysaccharide from a S. pneumoniae serotypeconjugated to a carrier protein, and wherein the up to 30 polysaccharideprotein conjugates include polysaccharides of a group of S. pneumoniaeserotypes selected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F,8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F,33F and 35B.
 29. The multivalent immunogenic composition of claim 28,wherein the up to 30 polysaccharide protein conjugates further includeone, two, three, four, five or six additional S. pneumoniae serotypesselected from 7C, 9N, 16F, 23A, 35F and
 38. 30. A multivalentimmunogenic composition comprising up to 30 distinct S. pneumoniaepolysaccharide protein conjugates, wherein each of the conjugatescomprises a polysaccharide from a S. pneumoniae serotype conjugated to acarrier protein, and wherein the up to 30 polysaccharide proteinconjugates include polysaccharides of a group of S. pneumoniae serotypesselected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V,10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and35B.
 31. The multivalent immunogenic composition of claim 30, whereinthe up to 30 polysaccharide protein conjugates further include one, two,three, four, five or six additional S. pneumoniae serotypes selectedfrom 7C, 9N, 16F, 23A, 35F and
 38. 32. The multivalent immunogeniccomposition of any of claims 28 to 31, wherein the carrier protein isCRM197.
 33. The multivalent immunogenic composition of claim 32, whereineach of the polysaccharide protein conjugates is formed by a conjugationreaction comprising an aprotic solvent, wherein the aprotic solvent isdimethylsulfoxide (DMSO).
 34. The multivalent immunogenic composition ofany of claims 28 to 33, wherein the composition comprises an adjuvant.